Methods of producing aggregate-free monomeric diphtheria toxin fusion proteins and therapeutic uses

ABSTRACT

The present invention is a DNA expression vector comprising: a toxP: a mutant toxO that blocks Fe-mediated regulation of gene expression; and a DNA sequence encoding a protein, wherein the toxP and the mutant toxO regulate expression of the DNA segment encoding the protein. It is preferred that DNA expression vectors of the present invention include DNA sequences encoding a signal peptide so that a protein expressed is attached to the signal peptide prior to processing. Novel proteins are produced off of the DNA expression vector of the present invention.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Divisional Application of U.S. application Ser.No. 16/611,243 filed Nov. 6, 2019, now pending; which is a 371 NationalStage Application of International Application No. PCT/US2017/051020filed Sep. 11, 2017, now expired and claims priority to InternationalApplication No. PCT/US2017/021715 filed Mar. 10, 2017; U.S. applicationSer. No. 16/611,243 is also a Continuation-in-Part Application of U.S.application Ser. No. 16/083,848 filed Sep. 10, 2018, now issued as U.S.Pat. No. 10,988,512; which is a 371 National Stage Application ofInternational Application No. PCT/US2017/021715 filed Mar. 10, 2017,which claims the benefit under 35 USC § 119(e) to U.S. Application Ser.No. 62/306,281 filed Mar. 10, 2016, now expired. The disclosure of eachof the prior applications is considered part of and is incorporated byreference in the disclosure of this application.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH R DEVELOPMENT

This invention was made with government support under Grant Nos.A1037856, A1036973, A1097138 and UC7A1095321-01 awarded by the NationalInstitutes of Health. The government has certain rights in theinvention.

INCORPORATION BY REFERENCE OF SEQUENCE LISTING

The material in the accompanying sequence listing is hereby incorporatedby reference into this application. The accompanying sequence listingtext file, named JHU4270-3_Seq.Listing.txt, was created on Dec. 13, 2021and is 140 kb in size. The file can be accessed using Microsoft Word ona computer that uses Windows OS.

BACKGROUND OF THE INVENTION

Ontak® (denileukin diftitox), is a 521 amino acid, recombinant,DNA-derived cytotoxic protein composed of the sequences for diphtheriatoxin fragments A and a portion of fragment B (Meti-His₃₈₈) and thesequences for human interleukin-2 (IL-2; Alai-Thri33). It is currentlyproduced in an E. coli expression system and has a molecular weight of58 kD. Neomycin is used in the fermentation process but is undetectablein the final product. Ontak®, which is supplied in single use vials as asterile, frozen solution intended for intravenous (IV) administration,was approved by the FDA in 1999 for the treatment of cutaneous T celllymphoma (CTCL). The FDA placed Ontak® on clinical hold in June 2011because of concerns regarding the presence of protein aggregates ofheterogeneous molecular weight, excess residual DNA, and excess residualdetergent in the final formulation. The production of Ontak® wasachieved by expressing the recombinant protein in the E. coli cytoplasm,and this expression system resulted in the recombinant protein forminglarge insoluble aggregates or so-called inclusion bodies comprised ofthe Ontak® polypeptide. In the current process of production, whichincludes denaturation and refolding of the inclusion body forms, proteinaggregates of heterogeneous molecular weight were still present in thefinal formulation. The presence of these aggregates in the purified formis a consequence of using E. coli-derived cytoplasmic inclusion bodiesas the source of the polypeptide and because of the intrinsichydrophobic nature of the toxin's transmembrane domain even in thepresence of Tween 20. Ontak® produced using this method will hereafterbe referred to as classic-Ontak® or c-Ontak®.

In addition, like all of the bacterial and plant toxins, c-Ontak®carries amino acid motifs that induce vascular leak syndrome (VLS).Approximately 30% of patients treated with c-Ontak® develop VLS symptomsranging from peripheral edema with rapid weight gain to hypoalbuminemiato pulmonary edema. What is needed are 1) a process enabling theproduction of Ontak-like proteins at high yields and purity, eliminatingaggregates in the final commercial product, and 2) modified Ontak-likeproteins with minimal VLS side-effects to provide safer drugs topatients.

SUMMARY OF THE INVENTION

One embodiment of the present invention is a DNA expression vectorcomprising: a toxP; a mutant toxO that blocks Fe-mediated regulation ofgene expression; and a DNA sequence encoding a protein, wherein the toxPand the mutant toxO regulate expression of the DNA segment encoding theprotein. It is preferred that DNA expression vectors of the presentinvention include DNA sequences encoding a signal peptide so that aprotein expressed off a DNA expression vector is attached to the signalpeptide that is typically cleaved off to form a mature protein. Thepreferred mutant toxO is SEQ ID NO: 1 and the preferred signal peptideis SEQ ID NO: 5. The DNA expression vectors of the present invention maybe used to produce many kinds of proteins such as CRM 197 and CRM 107,or a combination thereof. CRM protein sequences are illustrated in SEQID NOs: 18-21. It is preferred that the DNA expression vectors of thepresent invention encode a diphtheria toxin, or functional part thereof,attached to a receptor binding protein, or a functional part thereof toform a diphtheria toxin receptor fusion protein. The receptor bindingprotein portion of such fusion proteins may be selected from the groupcomprising IL-2, IL-3, IL-4, IL-6, IL-7, IL-15, EGF, FGF, substance P,CD4, αMSH, GRP, TT fragment C, GCSF, heregulin β1, a functional partthereof, or a combination thereof. Examples of diphtheria toxin fusionproteins include the proteins illustrated in any one of SEQ ID NOs:11-15, 30, 38-40, 42-43, 45-46, and 58, and proteins encoded by anucleic acid of any one of SEQ ID NOs: 31, 41, 44, and 59.

Another embodiment of the present invention is a DNA expression vectorcomprising: a toxP; a mutant toxO that blocks Fe-mediated regulation ofgene expression; a DNA sequence encoding a protein comprising a signalsequence, a diphtheria toxin, or a functional part thereof, that is freeof a diphtheria receptor binding domain or has a non-functionaldiphtheria toxin receptor binding domain, and a target receptor bindingdomain selected from the group comprising IL-2, IL-3, IL-4, IL-6, IL-7,IL-15, EGF, FGF, substance P, CD4, αMSH, GRP, TT fragment C, GCSF,heregulin β1, a functional part thereof, or a combination thereof,wherein the toxP and the mutant toxO regulate expression of the DNAsequence encoding the protein. Typically, a bacteria transformed with aDNA expression vector of the present invention produces a diphtheriatoxin receptor binding fusion protein attached to a signal peptide thatis directed to a periplasm, a culture medium, or both locations by thesignal peptide. If the bacteria is E. coli then the signal peptidetypically directs the diphtheria toxin receptor binding fusion proteinto the periplasm. If the bacteria is Corynebacterium diphtheria thensignal peptide directs the diphtheria toxin receptor binding fusionprotein to the culture medium. It is preferred that a DNA expressionvector of the present invention comprises SEQ ID NO: 3 and may comprisea DNA encoding a cleavable protein tag wherein the cleavable protein tagis attached to the diphtheria toxin receptor binding fusion protein.Example of diphtheria toxin receptor binding fusion proteins producedfrom the DNA expression vectors of the present invention include any oneof SEQ ID NOs: 11-15, 30, 38-40, 42-43, 45-46, and 58, and proteinsencoded by a nucleic acid of any one of SEQ ID NOs: 31, 41, 44, and 59.

Another embodiment of the present invention includes a method forproducing aggregate-free monomeric diphtheria toxin fusion proteinscomprising the following steps: transforming bacteria with a DNAexpression vectors of the present invention; forming a transformant;incubating the transformant in a culture medium to allow expression of aprotein that is secreted into the culture medium; and purifying theprotein from the culture medium. The preferred bacteria used in thismethod is Corynebacterium diphtheria.

Another embodiment of the present invention includes a method forproducing aggregate-free monomeric diphtheria toxin fusion proteinscomprising the following steps: 1) transforming Corynebacteriumdiphtheria strain with a DNA vector comprising: a toxP; a mutant toxOthat blocks Fe-mediated regulation of gene expression; a DNA sequenceencoding a protein comprising: signal peptide; a diphtheria toxin, or afunctional part thereof, that is free of a diphtheria receptor bindingdomain or has a non-functional diphtheria toxin receptor bindingdomain,; and a target receptor binding domain selected from the groupcomprising IL-2, IL-3, IL-4, IL-6, IL-7, IL-15, EGF, FGF, substance P,CD4, αMSH, GRP, TT fragment C, GCSF, heregulin β1, TNFα, TGFβ, afunctional part thereof, or a combination thereof, wherein the toxP andthe mutant toxO regulate expression of the DNA sequence encoding theprotein; 2) forming a transformant; 3) incubating the transformant in aculture medium to allow expression of the protein and that is secretedinto the culture medium; and 4) purifying the diphtheria toxin fusionprotein from the culture medium. Examples of diphtheria toxin receptorfusion proteins produced by methods of the present invention include anyone of SEQ ID NOs: 11-15, 30, 38-40, 42-43, 45-46, and 58, and proteinsencoded by a nucleic acid of any one of SEQ ID NOs: 31, 41, 44, and 59.The preferred Corynebacterium diphtheria strain used in the methods ofthe present invention is Corynebacterium C7 beta (−), tox (−).

Another embodiment of the present invention includes a method oftreating a patient with tuberculosis comprising the following steps:preparing a diphtheria toxin fusion protein as provided in thisapplication; administering the diphtheria toxin fusion protein to apatient with tuberculosis.

Another embodiment of the present invention includes a DNA expressionvector comprising a mutant toxO promoter.

Another embodiment of the present invention includes a Corynebacteriumdiphtheria strain containing a DNA expression vector of the presentinvention.

Another embodiment of the present invention is method of making aprotein comprising the following steps: providing a DNA expressionvector comprising a toxP, a mutant /OA) that blocks Fe-mediatedregulation of gene expression, a signal sequence, and a DNA sequenceencoding a protein; transforming a bacteria strain with the DNA vectorto form a transformant; incubating the transformant in a culture mediumfor a period of time to allow expression of a protein that is secretedinto the culture medium; and purifying the protein from the culturemedium.

Another embodiment of the present invention is a fusion protein selectedfrom any one of SEQ ID NOs: 11-15, 30, 38-40, 42-43, 45-46, and 58, orencoded by a nucleic acid of any one of SEQ ID NOs: 31, 41, 44, and 59.

Another embodiment of the present invention is a pharmaceuticalcomposition comprising a fusion protein described above.

Another embodiment of the present invention is a pharmaceuticalcomposition comprising a fusion protein describe above, and at least oneor more other chemotherapy agents. Examples of chemotherapy agentsinclude isoniazid, rifampin, rifabutin, rifapentine, pyrazinamide,ethambutol, streptomycin, amikacin, kanamycin, ethionamide,protionamide, terizidone, thiacetazone, cycloserine, capreomycin,para-amino salicylic acid (PAS), viomycin, ofloxacin, ciprofloxacin,levofloxacin, moxifloxacin, bedaquiline, or delamanid, linezolid,tedezolid, amoxicillin-clavulanic acid, meropenem, imipenem,clarithromycin or clofazimine.

A pharmaceutical composition of comprising a fusion protein describedabove, and at least one or more other antimicrobial agents. Examples ofantimicrobial agents include isoniazid, rifampin, rifabutin,rifapentine, pyrazinamide, ethambutol, streptomycin, amikacin,kanamycin, ethionamide, protionamide, terizidone, thiacetazone,cycloserine, capreomycin, para-amino salicylic acid (PAS), viomycin,ofloxacin, ciprofloxacin, levofloxacin, moxifloxacin, bedaquiline, ordelamanid, linezolid, tedezolid, amoxicillin-clavulanic acid, meropenem,imipenem, clarithromycin, or clofazimine.

Another embodiment of the present invention is a method of treating orpreventing cancer in a subject comprising administering to the subjectan effective amount of a pharmaceutical composition comprising a fusionprotein selected from any one of SEQ ID NOs: 11-15, 30, 38-40, 42-43,45-46, and 58, or encoded by a nucleic acid selected from any one of SEQID NOs: 31, 41, 44, and 59.

Another embodiment of the present invention is a method of treating orpreventing tuberculosis in a subject comprising administering to thesubject an effective amount of a pharmaceutical composition comprising afusion protein selected from any one of SEQ ID NOs: 11-15, 30, 38-40,42-43, 45-46, and 58, or encoded by a nucleic acid selected from any oneof SEQ ID NOs: 31, 41, 44, and 59.

Another embodiment of the present invention is a prokaryotic cell linecomprising a DNA expression vector of the present invention.

Another embodiment of the present invention is kit comprising the DNAexpression vector of the present invention.

Another embodiment of the present invention is a toxP comprising SEQ IDNO: 2.

Another embodiment of the present invention is a protein of any one ofSEQ ID NOs: 11-15, 30, 38-40, 42-43, 45-46, and 58, or a protein encodedby a nucleic acid selected from any one or SEQ ID NOs: 31, 41, 44, and59.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1a-1b illustrate: 1 a) a mutant toxO of the present invention (SEQID NO: 1) and 1 b) a wild type toxO (SEQ ID NO: 25) and a DtxR consensusbinding sequence.

FIGS. 2a-2b illustrate: 2 a) the classic denileukin diftitox(c-denileukin diftitox) expression vector used to manufacture Ontak® and2 b) the secreted denileukin diftitox (s-denileukin diftitox) expressionvector including the tox promoter (toxP), and mutant toxO of the presentinvention. FIG. 2a discloses SEQ ID NO: 26 and FIG. 2b discloses SEQ IDNO: 27.

FIG. 3 illustrates a vascular leak mutant (VLM) called c-denileukindiftitox-VLM has equivalent potency to c-denileukin diftitox in killingIL2-receptor-bearing cells.

FIG. 4 illustrates c-denileukin diftitox-VLM does not cause vascularleak in vitro.

FIG. 5 illustrates that c-denileukin diftitox-VLM has significantly lessacute toxicity in vivo than c-Ontak® using a mouse survival model.

FIG. 6 illustrates a diphtheria toxin-based fusion protein toxinplatform technology of the present invention.

FIG. 7 illustrates plasmid pKN2.6Z-LC127 with the tox promoter (toxP ofSEQ ID NO: 2) and a mutant tox operator (toxO) (DNA SEQ ID NO: 1), asignal peptide (DNA SEQ ID NO: 4) attached to c-denileukin diftitox DNAsequences (DNA SEQ ID NO: 6).

FIGS. 8a-8b illustrate: 8 a) the problems of the conventional process ofmanufacturing Ontak® as cytoplasmic inclusion bodies in E. coli and 8 b)illustrates easy and clean manufacturing process of producing asecreted-denileukm diftitox having one less amino acid than the Ontak®protein. FIG. 8a discloses “fMGADD” as SEQ ID NO: 28 and FIG. 8bdiscloses “GADD” as SEQ ID NO: 29.

FIG. 9 illustrates an immunoblot of s-denileukin diftitox prepared bythe process of the present invention where s-denileukin diftitox isexpressed within a Corynebacterium diphtheria strain C7 beta (−), tox(−) and is secreted into the culture medium.

FIG. 10 illustrates how a denileukin diftitox of the present invention,is expected to deplete IL-2R (CD25+) bearing T cells (T_(regs)) within atuberculosis granuloma. T_(regs) are immunosuppressive by theirinhibition of T_(eff) cells.

FIG. 11 illustrates diphtheria fusion proteins used in the in vivotreatment of subjects (mice) with M. tuberculosis.

FIG. 12 illustrates the results of treating subjects (mice) infectedwith M. tuberculosis with diphtheria toxin-based fusion proteins.

FIG. 13 illustrates a diphtheria toxin-based fusion protein regimen fortreating subjects (mice) infected with M. tuberculosis.

FIG. 14 illustrates the use of a diphtheria toxin-based fusion proteinto treat subjects (humans) with malignant melanoma.

FIG. 15 illustrates the three constructs for rapid production of VLMs-Ontak and related proteins using His (histidine tags) (“His₆/6x His”and “His₉/9x His” disclosed as SEQ ID NOs: 23 and 48, respectively).

FIG. 16 illustrates purified VLM s-Ontak-His₆ (“His₆” disclosed as SEQID NO: 23) at greater than 97% purity produced using the C-terminal His6VLM s-Ontak construct (“His₆” disclosed as SEQ ID NO: 23). Specifically,a recombinant C. diphtherias harboring a gene construct encoding VLMs-Ontak-His₆ (“His₆” disclosed as SEQ ID NO: 23) was grown to an opticaldensity (OD) of ˜12. The culture supernatant was harvested, concentratedby tangential flow filtration using a 30 kDa molecular weight cut-offmembrane, and diafiltered for buffer exchange using tangential flowfiltration as above. The protein mixture was partially purified byNi-affinity chromatography and then purified to greater than 97% by gelpermeation chromatography using S-100 resin. The resulting VLMs-Ontak-His₆ (“His₆” disclosed as SEQ ID NO: 23) was >97% pure.

FIG. 17 illustrates purified s-Ontak at greater than 97% purity producedusing the C-terminal His₆ s-Ontak construct (SEQ ID NOs: 58-59; “His₆”disclosed as SEQ ID NO: 23). Specifically, a recombinant C. diphtheriaeharboring a gene construct encoding s-Ontak-His₆ (“His₆” disclosed asSEQ ID NO: 23) was grow n to OD ˜12. The culture supernatant washarvested, concentrated by tangential flow filtration using a 30 kDamolecular weight cut-off membrane, and diafiltered for buffer exchangeusing tangential flow filtration as above. The protein mixture waspartially purified by Ni-affinity chromatography and then purified togreater than 97% by gel permeation chromatography using S-100 resin. Theresulting s-Ontak-His₆ (“His₆” disclosed as SEQ ID NO: 23) was >97% pureand stable at 4° C.

FIG. 18 illustrates purified VLM s-Ontak-His₆ (“His₆” disclosed as SEQID NO: 23) at greater than 97% purity produced using the C-terminal His₆VLM s-Ontak construct (SEQ ID NO: 23). Specifically, a recombinant C.diphtheriae harboring a gene construct encoding VLM s-Ontak-His₆ (“His₆”disclosed as SEQ ID NO: 23) was grown to OD ˜12. The culture supernatantwas harvested, concentrated by tangential flow filtration using a 30 kDamolecular weight cut-off membrane, and diafiltered for buffer exchangeusing tangential flow filtration as above. The protein mixture waspartially purified by Ni-affinity chromatography and then purified togreater than 97% by gel permeation chromatography using S-100 resin. Theresulting VLM s-Ontak-His₆ (“His₆” disclosed as SEQ ID NO: 23) was >97%pure.

FIG. 19 illustrates the S-100 gel filtration column used to purifys-Ontak-His₆ (“His₆” disclosed as SEQ ID NO: 23) and VLM s-Ontak-His₆(“His₆” disclosed as SEQ ID NO: 23) was calibrated for retention ofproteins of known molecular weight: g-globulin (158 kDa), ovalbumin(43.5 kDa), and myoglobin (17 kDa). The retention time for s-Ontak-His₆(“His₆” disclosed as SEQ ID NO: 23) was 94 minutes, confirming that thes-Ontak-His₆ polypeptide is a >97% aggregate-free, full-length,monomeric diphtheria toxin fusion protein with an apparent molecularweight of 58 kDa and neither dimers nor higher order aggregates weredetected by immunoblot probed with monoclonal anti-IL-2 antibody.

DEFINITIONS

Unless defined otherwise, all technical and scientific terms used hereinhave the meaning commonly understood by a person skilled in the art towhich this invention belongs. The following references provide one ofskill with a general definition of many of the terms used in thisinvention: Singleton et al., Dictionary of Microbiology and MolecularBiology (2nd ed. 1994); The Cambridge Dictionary of Science andTechnology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R.Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, TheHarper Collins Dictionary of Biology (1991). As used herein, thefollowing terms have the meanings ascribed to them below, unlessspecified otherwise.

The term “activity” refers to the ability of a gene to perform itsfunction such as Indoleamine 2,3-dioxygenase (an oxidoreductase)catalyzing the degradation of the essential amino acid tryptophan (trp)to N-formyl-kynurenine.

By “agent” is meant any small molecule chemical compound, antibody,nucleic acid molecule, or polypeptide, or fragments thereof.

By “ameliorate” is meant decrease, suppress, attenuate, diminish,arrest, or stabilize the development or progression of a disease.

By “alteration” is meant a change (increase or decrease) in theexpression levels or activity of a gene or polypeptide as detected bystandard art known methods such as those described herein. As usedherein, an alteration includes a 10% change in expression levels,preferably a 25% change, more preferably a 40% change, and mostpreferably a 50% or greater change in expression levels.

By “analog” is meant a molecule that is not identical, but has analogousfunctional or structural features. For example, a polypeptide analogretains the biological activity of a corresponding naturally-occurringpolypeptide, while having certain biochemical modifications that enhancethe analog's function relative to a naturally occurring polypeptide.Such biochemical modifications could increase the analog's proteaseresistance, membrane permeability, or half-life, without altering, forexample, ligand binding. An analog may include an unnatural amino acid.

c- means “classic” when attached to a term such as c-denileukin diftitoxmeans Ontak® or that commercially available protein.

By “disease” is meant any condition or disorder that damages orinterferes with the normal function of a cell, tissue, or organ.Examples of diseases include cancer and tuberculosis.

By “effective amount” is meant the amount of a required to amelioratethe symptoms of a disease relative to an untreated patient. Theeffective amount of active compound(s) used to practice the presentinvention for therapeutic treatment of a disease varies depending uponthe manner of administration, the age, body weight, and general healthof the subject. Ultimately, the attending physician or veterinarian willdecide the appropriate amount and dosage regimen. Such amount isreferred to as an “effective” amount.

The term “express” refers to the ability of a gene to express the geneproduct including for example its corresponding mRNA or proteinsequence(s).

By “fragment” is meant a portion of a polypeptide or nucleic acidmolecule. This portion contains, preferably, at least 10%, 20%, 30%,40%, 50%, 60%, 70%, 80%, or 90% of the entire length of the referencenucleic acid molecule or polypeptide. A fragment may contain 10, 20, 30,40, 50, 60, 70, 80, 90, or 100, 200, 300, 400, 500, 600, 700, 800, 900,or 1000 nucleotides or amino acids.

is—means “immature secreted” when attached to a term such asis-denileukm diftitox means immature secreted denileukin diftitox thatcontains a signal peptide.

n—means “mature secreted” when attached to a term such as ms-denileukindiftitox means mature secreted denileukin diftitox that has beenprocessed and no longer contains a signal peptide.

n—means “new” when attached to a term such as n-denileukin diftitoxmeans new denileukin diftitox.

As used herein, the terms “prevent,” “preventing,” “prevention,”“prophylactic treatment” and the like refer to reducing the probabilityof developing a disorder or condition in a subject, who does not have,but is at risk of or susceptible to developing a disorder or condition.

The terms “polypeptide,” “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidue is an analog or mimetic of a corresponding naturally occurringamino acid, as well as to naturally occurring amino acid polymers.Polypeptides can be modified, e.g., by the addition of carbohydrateresidues to form glycoproteins. The terms “polypeptide,” “peptide” and“protein” include glycoproteins, as well as non-glycoproteins.

The term “purity” refers to the amount of polypeptide of the inventionpresent in a pharmaceutical composition free of other polypeptides. Forexample, a polypeptide of the invention present in a pharmaceuticalcomposition having a purity of about 80% means that greater than about80% of polypeptide is full-length and contaminated by less than about20% of either product-related or unrelated polypeptides. Purity can bedetermined, for example, by SDS polyacrylamide gel electrophoresis andstaining with Coomassie blue, methods which are described in thisapplication or by other methods known to those skilled in the art.

The term “aggregate-free, full-length, monomeric polypeptide” refers tothe amount of polypeptide of the invention present in a pharmaceuticalcomposition in monomeric form. For example, a pharmaceutical compositionof the invention comprising greater than about 80% aggregate-free,full-length, monomeric polypeptide means that greater than about 80% ofthe full-length polypeptide is in monomeric form. The amount ofaggregate-free, full-length, monomeric polypeptide can be determined,for example, by gel permeation chromatography using known monomericpolypeptides as size standards or by non-reducing, SDS-free nativepolyacrylamide gel electrophoresis, methods which are described in thisapplication or by other methods known to those skilled in the art.

By “reduces” is meant a negative alteration of at least 10%, 25%, 50%,75%, or 100%.

A “reference” refers to a standard or control conditions such as asample (human cells) or a subject that is a free, or substantially free,of an agent such as one or more compositions of the present inventioncomprising a nucleic acid or protein sequence such as anyone of SEQ IDNOs: 11-15, or fusion proteins thereof

A “reference sequence” is a defined sequence used as a basis forsequence comparison. A reference sequence may be a subset of or theentirety of a specified sequence; for example, a segment of afull-length cDNA or gene sequence, or the complete cDNA or genesequence. For polypeptides, the length of the reference polypeptidesequence will generally be at least about 16 amino acids, preferably atleast about 20 amino acids, more preferably at least about 25 aminoacids, and even more preferably about 35 amino acids, about 50 aminoacids, or about 100 amino acids. For nucleic acids, the length of thereference nucleic acid sequence will generally be at least about 50nucleotides, preferably at least about 60 nucleotides, more preferablyat least about 75 nucleotides, and even more preferably about 100nucleotides or about 300 nucleotides or any integer thereabout or therebetween.

s—means “secreted” when attached to a term such as s-denileukin diftitoxmeans secreted denileukin diftitox. Secreted denileukin diftitoxincludes is- and m-forms.

As used herein, the term “subject” is intended to refer to anyindividual or patient to which the method described herein is performed.Generally the subject is human, although as will be appreciated by thosein the art, the subject may be an animal. Thus other animals, includingmammals such as rodents (including mice, rats, hamsters and guineapigs), cats, dogs, rabbits, farm animals including cows, horses, goats,sheep, pigs, etc., and primates (including monkeys, chimpanzees,orangutans and gorillas) are included within the definition of subject.

By “substantially identical” is meant a polypeptide or nucleic acidmolecule exhibiting at least 50% identity to a reference amino acidsequence (for example, any one of the amino acid sequences describedherein) or nucleic acid sequence (for example, any one of the nucleicacid sequences described herein). Preferably, such a sequence is atleast 60%, more preferably 80% or 85%, and more preferably 90%, 95% oreven 99% identical at the amino acid level or nucleic acid to thesequence used for comparison.

Sequence identity is typically measured using sequence analysis software(for example, Sequence Analysis Software Package of the GeneticsComputer Group, University of Wisconsin Biotechnology Center, 1710University Avenue, Madison, Wis. 53705, BLAST, BESTFIT, GAP, orPILEUP/PRETTYBOX programs). Such software matches identical or similarsequences by assigning degrees of homology to various substitutions,deletions, and/or other modifications. Conservative substitutionstypically include substitutions within the following groups: glycine,alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid,asparagine, glutamine; serine, threonine; lysine, arginine; andphenylalanine, tyrosine. In an exemplary approach to determining thedegree of identity, a BLAST program may be used, with a probabilityscore between e⁻³ and e⁻¹⁰⁰ indicating a closely related sequence.

Ranges provided herein are understood to be shorthand for all of thevalues within the range. For example, a range of 1 to 50 is understoodto include any number, combination of numbers, or sub-range from thegroup consisting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.

As used herein, the terms “treat,” “treating,” “treatment,” and the likerefer to reducing or ameliorating a disorder and/or symptoms associatedtherewith. It will be appreciated that, although not precluded, treatinga disorder or condition does not require that the disorder, condition orsymptoms associated therewith be completely eliminated.

Unless specifically stated or obvious from context, as used herein, theterm “or” is understood to be inclusive. Unless specifically stated orobvious from context, as used herein, the terms “a”, “an”, and “the” areunderstood to be singular or plural.

Unless specifically stated or obvious from context, as used herein, theterm “about” is understood as within a range of normal tolerance in theart, for example within 2 standard deviations of the mean. About can beunderstood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%,0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear fromcontext, all numerical values provided herein are modified by the termabout.

The recitation of a listing of chemical groups in any definition of avariable herein includes definitions of that variable as any singlegroup or combination of listed groups. The recitation of an embodimentfor a variable or aspect herein includes that embodiment as any singleembodiment or in combination with any other embodiments or portionsthereof.

Any compositions or methods provided herein can be combined with one ormore of any of the other compositions and methods provided herein.

VLM—means “vascular leakage mutant” when attached to a tem such asdenileukin diftitox-VLM means denileukin diftitox vascular leakagemutant.

w—means “wild type” when attached to a term such as w-diphtheria toxinmeans wild type-diphtheria toxin.

DETAILED DESCRIPTION OF THE INVENTION

One embodiment of the present invention is the discovery of a processthat produces aggregate-free, monomeric, diphtheria toxin fusionproteins having enhanced purity and quality.

This process includes transforming bacteria including preferably,strains of Corynebacterium diphtheria with DNA expression vectors of thepresent invention. DNA expression vectors of the present invention aredesigned to include specific genetic elements comprising a tox promoter(toxP) and an overlapping novel, mutated tox operator (toxO), preferablya signal sequence, and a DNA sequence encoding a protein. The protein ispreferably a fusion protein including a diphtheria toxin, or functionalpart thereof, and a target receptor binding domain or a functional partthereof. The term “functional part thereof” means a part of a diphtheriatoxin protein that acts as a toxin or the part of a target receptorbinding domain that binds to its receptor. DNA expression vectors of thepresent invention are designed so proteins are expressed from a toxpromoter (toxP) and a mutant tox operator (toxO).

Mutant toxO

toxO, is a 19-bp operator region that is composed of two 9 bp imperfectpalindromic arms interrupted by a central cytosine (C) base. The wildtype toxO (FIG. 1b ) and a mutant toxO (FIG. 1a ) operator discovered byinventors are shown in FIG. 1. SEQ ID NO: 1 illustrates one embodimentof the DNA sequence of a mutant toxO this invention. toxP is a promoterhaving a DNA sequence of SEQ ID NO: 2. SEQ ID NO: 2 illustrates the toxPDNA sequences include the toxO DNA sequences. SEQ ID NO: 3 is a DNAsequence including a toxP, a toxO, a signal peptide, and a DNA sequenceencoding a protein. The asterisks in SEQ ID NO: 3 indicate the changesintroduced to create the mutant toxO.

(Mutant toxO DNA sequence) SEQ ID NO: 1 TTAGGATAGCTAAGTCCAT(toxP including the mutant toxO DNA sequencewhere the mutant toxO sequence is underlined) SEQ ID NO: 2TTGATTTCAGAGCACCCTTATAATTAGGATAGCTAAGTCCAT

The toxO DNA operator sequence is bound by a protein known as thediphtheria toxin repressor, DtxR. DtxR is a global iron-activatedregulatory protein that is able to control gene expression. Iniron-replete conditions, Fe²⁺ and Fe³⁺ ions bind to apo-DtxR causing aconformational change that allows the formation of homodimers of theDtxR repressor, which bind to the tox operator (toxO) DNA sequence andrepress tox gene expression. In low iron environments, Fe²⁺ and Fe³⁺ions disassociate from DtxR causing it to lose its DNA bindingcapability and disassociate from the operator; this event thereby allowsexpression of tox gene products. FIG. 1b illustrates the wild type toxODNA sequence.

To overcome the inhibitory effect of Fe²⁺ and Fe³⁺ ions on toxexpression, a DNA expression vector was created replacing the wild type(WT) toxO with a mutant toxO DNA sequence. This change blocks Feion-mediated regulation of tox gene expression. FIG. 1a , SEQ ID NO: 1,and SEQ ID NO: 3 illustrate the mutant /OA) DNA sequence of the presentinvention. Under this invention, bacteria such as E. coli and C.diphtheria harboring a recombinant plasmid encoding a diphtheria toxinfusion protein under the control of toxP and the mutant toxO may begrown in Fe-replete media, allowed to grow to high densities, and willnot require a shift to Fe-free media to induce expression. Theconstitutive expression of tox gene products in iron replete mediumrepresents a significant advance in the field. C. diphtheria,specifically the C7 beta (−), tox (−) strain is the preferred hostbacteria for the production of all diphtheria-toxin related recombinantproteins using the DNA expression vectors of the present invention. TheDNA expression vectors of the present invention may be used in otherbacteria such as E. coli.

DNA Expression Vectors

The DNA expression vectors of the present invention includes a toxP,mutant toxO, a DNA sequence encoding a protein, and preferably a signalsequence. SEQ ID NO: 3 is one example of a DNA sequence containing thesegenetic elements that may be part of a DNA expression vector of thepresent invention. As mentioned, the asterisks observed in SEQ ID NO: 3are placed above the base pair changes between the mutant and wild typetoxO. SEQ ID NO: 3 is numbered such that the toxP extends from base 1 to30, and toxO begins at base 24 and ends at base 42 (prior to theunderlined DNA sequence). The underlined DNA sequence represents base 74to base 148 and is a region of DNA encoding a 25 amino acid signalsequence (also observe in SEQ ID NO: 4, SEQ ID NO: 5, and FIG. 2). TheDNA expression vectors of the present invention are preferablyconstructed so one or more proteins are expressed from toxP, mutanttoxO, and are translated with an N-terminal signal sequence. TheN-terminal signal sequence targets the one or more proteins (expressedfrom the vector) for secretion, and the N-terminal signal peptide islater cleaved to make mature active proteins. SEQ ID NO: 3 includes DNAsequences encoding proteins such as a novel denileukin diftitox calledsecreted-denileukin diftitox, or s-denileukin diftitox. The s-denileukindiftitox has two forms called immature secreted-denileukin diftitox(is-denileukin diftitox) and mature secreted-denileukin diftitox(ms-denileukin diftitox). SEQ ID NO: 12 is of is-denileukin diftitox ofthe present invention and SEQ ID NO: 13 is of ms-denileukm diftitox ofthe present invention. The is-denileukin diftitox contains a signalsequence that during processing is cleaved off to form ms-denileukindiftitox. In addition, SEQ ID NO: 3 includes a DNA sequence beginning atbase 149 to 1711 that encodes a protein, specifically a fusion proteincontaining the functional parts of a diphtheria toxin and the functionalparts of IL 2. A new denileukin diftitox fusion protein sequence isformed called ms-denileukin diftitox that is a 520 amino acidpolypeptide and is composed of the ammo acid sequences for diphtheriatoxin fragments A and a portion of fragment B (Gly₁-His₃₈₇) and thesequences for human interleukin-2. As a result of cleavage of the signalsequence, ms-denileukin diftitox of the present invention lacks thefirst methionine present in classic-denileukin diftitox (c-denileukindiftitox) and is thereby one amino acid shorter than the amino acidsequence of the classic-denileukin diftitox protein known as Ontak®. SEQID NO: 13 is the protein sequence of the new diftitox protein sequencems-denileukm diftitox which may be compared to SEQ ID NO: 10 containingthe protein sequence of the classis-denileukin diftitox (c-denileukindiftitox) known as Ontak®.

DNA expression vectors of the present invention include DNA sequencesencoding one or more protein(s). A preferred protein of the presentinvention is a fusion protein comprising a diphtheria toxin (or afunctional part thereof) and a target receptor binding protein (or afunctional part thereof). An example of a diphtheria toxin that may beproduced from a DNA expression is any functional part of a diphtheriatoxin or any functional part of a diphtheria toxin vascular leakagemutant. Examples of proteins of target receptor binding domains producedfrom a DNA expression vector of the present invention include, IL-2,IL-3, IL-4, IL-6, IL-7, IL-15, EGF, FGF, substance P, CD4, αMSH, GRP, TTfragment C, GCSF, heregulin β1, TNFα, TGFβ, or a combination thereof.Other target receptor binding domains may be used depending upon thetherapeutic application; however, SEQ. ID NO. 9 is a preferred DNAsequence encoding a functional part of IL2 receptor binding domain. Forthe purposes of the present invention, some of the DNA plasmids and thegenetic elements thereof are illustrated in FIG. 1, FIG. 2, FIG. 6, andFIG. 7. Examples of fusion proteins encoded by DNA expression vectors ofthe present invention include SEQ ID NOs: 11, 12, 13, 14, 15, 19, and21.

(DNA sequence encoding secreted-denileukin diftitox ors-denileukin diftitox Sequence includes toxP, mutanttoxO, signal sequence, a functional part of diphtheriatoxin and a functional part of IL2. Bold font and asterisks indicate the changes introduces to create  the mutant toxO).SEQ ID NO: 3                                         **** * *   1  TTGATTTCAGAGCACCCTTATAATTAGGATAGCTAAGTCCATTATTTTAT  51  GAGTCCTGGTAAGGGGATACGTTGTGAGCAGAAAACTGTTTGCGTCAATC 101  TTAATAGGGGCGCTACTGGGGATAGGGGCCCCACCTTCAGCCCATGCAGG 151  CGCTGATGATGTTGTTGATTCTTCTAAATCTTTTGTGATGGAAAACTTTT 201  CTTCGTACCACGGGACTAAACCTGGTTATGTAGATTCCATTCAAAAAGGT 251  ATACAAAAGCCAAAATCTGGTACACAAGGAAATTATGACGATGATTGGAA 301  AGGGTTTTATAGTACCGACAATAAATACGACGCTGCGGGATACTCTGTAG 351  ATAATGAAAACCCGCTCTCTGGAAAAGCTGGAGGCGTGGTCAAAGTGACG 401  TATCCAGGACTGACGAAGGTTCTCGCACTAAAAGTGGATAATGCCGAAAC 451  TATTAAGAAAGAGTTAGGTTTAAGTCTCACTGAACCGTTGATGGAGCAAG 501  TCGGAACGGAAGAGTTTATCAAAAGGTTCGGTGATGGTGCTTCGCGTGTA 551  GTGCTCAGCCTTCCCTTCGCTGAGGGGAGTTCTAGCGTTGAATATATTAA 601  TAACTGGGAACAGGCGAAAGCGTTAAGCGTAGAACTTGAGATTAATTTTG 651  AAACCCGTGGAAAACGTGGCCAAGATGCGATGTATGAGTATATGGCTCAA 701  GCCTGTGCAGGAAATCGTGTCAGGCGATCAGTAGGTAGCTCATTGTCATG 751  CATCAACCTGGATTGGGATGTTATCCGTGATAAAACTAAAACTAAGATCG 801  AATCTCTGAAAGAACACGGTCCGATCAAAAACAAAATGAGCGAAAGCCCG 851  AACAAAACTGTATCTGAAGAAAAAGCTAAACAGTACCTGGAAGAATTCCA 901  CCAGACTGCACTGGAACACCCGGAACTGTCTGAACTTAAGACCGTTACTG 951  GTACCAACCCGGTATTCGCTGGTGCTAACTACGCTGCTTGGGCAGTAAAC1001  GTTGCTCAGGTTATCGATAGCGAAACTGCTGATAACCTGGAAAAAACTAC1051  CGCGGCTCTGTCTATCCTGCCGGGTATCGGTAGCGTAATGGGCATCGCAG1101  ACGGCGCCGTTCACCACAACACTGAAGAAATCGTTGCACAGTCTATCGCT1151  CTGAGCTCTCTGATGGTTGCTCAGGCCATCCCGCTGGTAGGTGAACTGGT1201  TGATATCGGTTTCGCTGCATACAACTTCGTTGAAAGCATCATCAACCTGT1251  TCCAGGTTGTTCACAACTCTTACAACCGCCCGGCTTACTCTCCGGGTCAC1301  AAGACGCATGCACCTACTTCTAGCTCTACCAAGAAAACCCAGCTGCAGCT1351  CGAGCACCTGCTGCTGGATTTGCAGATGATCCTGAACGGTATCAACAATT1401  ACAAGAACCCGAAACTGACGCGTATGCTGACCTTCAAGTTCTACATGCCG1451  AAGAAGGCCACCGAACTGAAACACCTGCAGTGTCTAGAAGAAGAACTGAA1501  ACCGCTGGAGGAAGTTCTGAACCTGGCTCAGTCTAAAAACTTCCACCTGC1551  GGCCGCGTGACCTGATCTCTAACATCAACGTAATCGTTCTGGAACTGAAG1601  GGCTCTGAAACCACCTTCATGTGTGAATACGCTGATGAGACCGCAACCAT1651  CGTAGAATTCCTGAACCGTTGGATCACCTTCTGTCAGTCTATCATCTCTA1701  CCCTGACCTGA < 1711 (Signal DNA Sequence) SEQ ID NO: 4  74  GTGAGCAGAAAACTGTTTGCGTCAATCTTAATAGGGGCGCTACTGGGGAT 124  AGGGGCCCCACCTTCAGCCCATGCA < 148 (Signal Protein Sequence)SEQ ID NO: 5  -25  MSRKLFASILIGALLGIGAPPSAHA < -1(classic-denileukin diftitox DNA sequence) SEQ ID NO: 6    1  ATG   4  GGCGCTGATGATGTTGTTGATTCTTCTAAATCTTTTGTGATGGAAAACTT  54  TTCTTCGTACCACGGGACTAAACCTGGTTATGTAGATTCCATTCAAAAAG 104  GTATACAAAAGCCAAAATCTGGTACACAAGGAAATTATGACGATGATTGG 154  AAAGGGTTTTATAGTACCGACAATAAATACGACGCTGCGGGATACTCTGT 204  AGATAATGAAAACCCGCTCTCTGGAAAAGCTGGAGGCGTGGTCAAAGTGA 254  CGTATCCAGGACTGACGAAGGTTCTCGCACTAAAAGTGGATAATGCCGAA 304  ACTATTAAGAAAGAGTTAGGTTTAAGTCTCACTGAACCGTTGATGGAGCA 354  AGTCGGAACGGAAGAGTTTATCAAAAGGTTCGGTGATGGTGCTTCGCGTG 404  TAGTGCTCAGCCTTCCCTTCGCTGAGGGGAGTTCTAGCGTTGAATATATT 454  AATAACTGGGAACAGGCGAAAGCGTTAAGCGTAGAACTTGAGATTAATTT 504  TGAAACCCGTGGAAAACGTGGCCAAGATGCGATGTATGAGTATATGGCTC 554  AAGCCTGTGCAGGAAATCGTGTCAGGCGATCAGTAGGTAGCTCATTGTCA 604  TGCATCAACCTGGATTGGGATGTTATCCGTGATAAAACTAAAACTAAGAT 654  CGAATCTCTGAAAGAACACGGTCCGATCAAAAACAAAATGAGCGAAAGCC 704  CGAACAAAACTGTATCTGAAGAAAAAGCTAAACAGTACCTGGAAGAATTC 754  CACCAGACTGCACTGGAACACCCGGAACTGTCTGAACTTAAGACCGTTAC 804  TGGTACCAACCCGGTATTCGCTGGTGCTAACTACGCTGCTTGGGCAGTAA 854  ACGTTGCTCAGGTTATCGATAGCGAAACTGCTGATAACCTGGAAAAAACT 904  ACCGCGGCTCTGTCTATCCTGCCGGGTATCGGTAGCGTAATGGGCATCGC 954  AGACGGCGCCGTTCACCACAACACTGAAGAAATCGTTGCACAGTCTATCG1004  CTCTGAGCTCTCTGATGGTTGCTCAGGCCATCCCGCTGGTAGGTGAACTG1054  GTTGATATCGGTTTCGCTGCATACAACTTCGTTGAAAGCATCATCAACCT1104  GTTCCAGGTTGTTCACAACTCTTACAACCGCCCGGCTTACTCTCCGGGTC1154  ACAAGACGCATGCACCTACTTCTAGCTCTACCAAGAAAACCCAGCTGCAG1204  CTCGAGCACCTGCTGCTGGATTTGCAGATGATCCTGAACGGTATCAACAA1254  TTACAAGAACCCGAAACTGACGCGTATGCTGACCTTCAAGTTCTACATGC1304  CGAAGAAGGCCACCGAACTGAAACACCTGCAGTGTCTAGAAGAAGAACTG1354  AAACCGCTGGAGGAAGTTCTGAACCTGGCTCAGTCTAAAAACTTCCACCT1404  GCGGCCGCGTGACCTGATCTCTAACATCAACGTAATCGTTCTGGAACTGA1454  AGGGCTCTGAAACCACCTTCATGTGTGAATACGCTGATGAGACCGCAACC1504  ATCGTAGAATTCCTGAACCGTTGGATCACCTTCTGTCAGTCTATCATCTC1554  TACCCTGACCTGA < 1566

Formation of Diphtheria Toxin Fusion Proteins Having Minimal, or no,Vascular Leakage (Denileukin Diftitox-VLMs)

Like all of the bacterial and plant toxins, denileukin diftitox carriesamino acid motifs that may induce vascular leak syndrome (VLS).Approximately 30% of patients treated with Ontak® develop VLS rangingfrom rapid weight gain with peripheral edema to hypoalbuminemiatopulmonary edema. Mutations were made to the DNA sequence of Ontak® asdescribed in U.S. Pat. No. 8,865,866. It was discovered that DNAmutations made to the DNA sequence such that the valine (GTT) at the7^(th) residue of SEQ ID NO: 10 is replaced with an alanine as shown inSEQ ID NO: 16, resulted in the fusion toxin having little, or no,vascular leak syndrome side effects. These mutants are referred to as“vascular leak mutants” (VLM). The vascular leak mutants, or denileukindiftitox-VLMS are shown to have the same potency as c-denileukindiftitox in FIG. 3, not to cause vascular leak in FIG. 4, and to havesignificantly less acute toxicity in vivo than c-denileukin diftitox inFIG. 5. s-denileukin diftitox-VLM, has an alanine replacing the valineat the 6^(th) residue shown in in SEQ ID NOs: 14 and 15. s-denileukindiftitox-VLM protein should have a similar decrease in toxicity as thatfound with the c-denileukin diftitox-VLM protein.

Also, the sequences V₂₉D₃₀S₃₁ and I₂₉₀D₂₉₁S₂₉₂ shown in SEQ ID NO: 10(ammo acid sequence of c-denileukin diftitox), when mutated also willreduce VLS. A claim in this discovery is that introduction ofsubstitutions in V₂₉D₃₀S₃₁ and/or I₂₉₀D₂₉₁S₂₉₂ such as V29A or 1290A maybe introduced into the corresponding positions of diphtheria toxinfusion proteins and that these substitutions will also have value infurther reducing vascular leakage syndrome.

(denileukin diftitox-VLM underlined codon encodes for alanine, here shown as GCT, described in  U.S. Pat. No. 8,865,866.)SEQ ID NO: 7    1  ATG   4  GGCGCTGATGATGTTGCTGATTCTTCTAAATCTTTTGTGATGGA       AAACTT  54  TTCTTCGTACCACGGGACTAAACCTGGTTATGTAGATTCCATTC       AAAAAG 104  GTATACAAAAGCCAAAATCTGGTACACAAGGAAATTATGACGAT       GATTGG 154  AAAGGGTTTTATAGTACCGACAATAAATACGACGCTGCGGGATA       CTCTGT 204  AGATAATGAAAACCCGCTCTCTGGAAAAGCTGGAGGCGTGGTCA       AAGTGA 254  CGTATCCAGGACTGACGAAGGTTCTCGCACTAAAAGTGGATAAT       GCCGAA 304  ACTATTAAGAAAGAGTTAGGTTTAAGTCTCACTGAACCGTTGAT       GGAGCA 354  AGTCGGAACGGAAGAGTTTATCAAAAGGTTCGGTGATGGTGCTT       CGCGTG 404  TAGTGCTCAGCCTTCCCTTCGCTGAGGGGAGTTCTAGCGTTGAA       TATATT 454  AATAACTGGGAACAGGCGAAAGCGTTAAGCGTAGAACTTGAGAT       TAATTT 504  TGAAACCCGTGGAAAACGTGGCCAAGATGCGATGTATGAGTATA       TGGCTC 554  AAGCCTGTGCAGGAAATCGTGTCAGGCGATCAGTAGGTAGCTCA       TTGTCA 604  TGCATCAACCTGGATTGGGATGTTATCCGTGATAAAACTAAAAC       TAAGAT 654  CGAATCTCTGAAAGAACACGGTCCGATCAAAAACAAAATGAGCG       AAAGCC 704  CGAACAAAACTGTATCTGAAGAAAAAGCTAAACAGTACCTGGAA       GAATTC 754  CACCAGACTGCACTGGAACACCCGGAACTGTCTGAACTTAAGAC       CGTTAC 804  TGGTACCAACCCGGTATTCGCTGGTGCTAACTACGCTGCTTGGG       CAGTAA 854  ACGTTGCTCAGGTTATCGATAGCGAAACTGCTGATAACCTGGAA       AAAACT 904  ACCGCGGCTCTGTCTATCCTGCCGGGTATCGGTAGCGTAATGGG       CATCGC 954  AGACGGCGCCGTTCACCACAACACTGAAGAAATCGTTGCACAGT       CTATCG1004  CTCTGAGCTCTCTGATGGTTGCTCAGGCCATCCCGCTGGTAGGT       GAACTG1054  GTTGATATCGGTTTCGCTGCATACAACTTCGTTGAAAGCATCAT       CAACCT1104  GTTCCAGGTTGTTCACAACTCTTACAACCGCCCGGCTTACTCTC       CGGGTC1154  ACAAGACGCATGCACCTACTTCTAGCTCTACCAAGAAAACCCAG       CTGCAG1204  CTCGAGCACCTGCTGCTGGATTTGCAGATGATCCTGAACGGTAT       CAACAA1254  TTACAAGAACCCGAAACTGACGCGTATGCTGACCTTCAAGTTCT       ACATGC1304  CGAAGAAGGCCACCGAACTGAAACACCTGCTGCAGTGTCTAGAA       GAAGAA1354  CTGAAACCGCTGGAGGAAGTTCTGAACCTGGCTCAGTCTAAAAA       CTTCCA1404  CCTGCGGCCGCGTGACCTGATCTCTAACATCAACGTAATCGTTC       TGGAAC1454  TGAAGGGCTCTGAAACCACCTTCATGTGTGAATACGCTGATGAG       ACCGCA1504  ACCATCGTAGAATTCCTGAACCGTTGGATCACCTTCTGTCAGTC       TATCAT1554  CTCTACCCTGACC < 1566

Alignment of DNA sequences comparing SEQ ID NO: 7 [denileukindiftitox-VLM described in US patent No. 8,865,866] with SEQ ID NO: 8[is-denileukin diftitox-VLM of the present invention] demonstrates SEQID NO: 8 is missing a codon (three bases) in line 1381-1437.

Similarity: 1563/1638 (95.42%)NO: 7     1  ------------------------------------------------------------     0             ############################################################NO: 8     1  GTGAGCAGAAAACTGTTTGCGTCAATCTTAATAGGGGCGCTACTGGGGATAGGGGCCCCA    60NO: 7     1  ----------ATG--GGCGCTGATGATGTTGCTGATTCTTCTAAATCTTTTGTGATGGAA    48             ##########|||## |||||||||||||||||||||||||||||||||||||||| |||NO: 8    61  CCTTCAGCCCATGCAGGCGCTGATGATGTTGCTGATTCTTCTAAATCTTTTGTGATGGAA   120NO: 7    49  AACTTTTCTTCGTACCACGGGACTAAACCTGGTTATGTAGATTCCATTCAAAAAGGTATA   108             ||||||||||||||| |||||||||||||||||||||||||||||||||||||||| |||NO: 8   121  AACTTTTCTTCGTACCACGGGACTAAACCTGGTTATGTAGATTCCATTCAAAAAGGTATA   180NO: 7   109  CAAAAGCCAAAATCTGGTACACAAGGAAATTATGACGATGATTGGAAAGGGTTTTATAGT   168             ||||||||||||||| |||||||||||||||||||||||||||||||||||||||| |||NO: 8   181  CAAAAGCCAAAATCTGGTACACAAGGAAATTATGACGATGATTGGAAAGGGTTTTATAGT   240NO: 7   169  ACCGACAATAAATACGACGCTGCGGGATACTCTGTAGATAATGAAAACCCGCTCTCTGGA   228             ||||||||||||||| |||||||||||||||||||||||||||||||||||||||| |||NO: 8   241  ACCGACAATAAATACGACGCTGCGGGATACTCTGTAGATAATGAAAACCCGCTCTCTGGA   300NO: 7   229  AAAGCTGGAGGCGTGGTCAAAGTGACGTATCCAGGACTGACGAAGGTTCTCGCACTAAAA   288             ||||||||||||||| |||||||||||||||||||||||||||||||||||||||| |||NO: 8   301  AAAGCTGGAGGCGTGGTCAAAGTGACGTATCCAGGACTGACGAAGGTTCTCGCACTAAAA   360NO: 7   289  GTGGATAATGCCGAAACTATTAAGAAAGAGTTAGGTTTAAGTCTCACTGAACCGTTGATG   348             ||||||||||||||| |||||||||||||||||||||||||||||||||||||||| |||NO: 8   361  GTGGATAATGCCGAAACTATTAAGAAAGAGTTAGGTTTAAGTCTCACTGAACCGTTGATG   420NO: 7   349  GAGCAAGTCGGAACGGAAGAGTTTATCAAAAGGTTCGGTGATGGTGCTTCGCGTGTAGTG   408             ||||||||||||||| |||||||||||||||||||||||||||||||||||||||| |||NO: 8   421  GAGCAAGTCGGAACGGAAGAGTTTATCAAAAGGTTCGGTGATGGTGCTTCGCGTGTAGTG   480NO: 7   409  CTCAGCCTTCCCTTCGCTGAGGGGAGTTCTAGCGTTGAATATATTAATAACTGGGAACAG   468             ||||||||||||||| |||||||||||||||||||||||||||||||||||||||| |||NO: 8   481  CTCAGCCTTCCCTTCGCTGAGGGGAGTTCTAGCGTTGAATATATTAATAACTGGGAACAG   540NO: 7   469  GCGAAAGCGTTAAGCGTAGAACTTGAGATTAATTTTGAAACCCGTGGAAAACGTGGCCAA   528             ||||||||||||||| |||||||||||||||||||||||||||||||||||||||| |||NO: 8   541  GCGAAAGCGTTAAGCGTAGAACTTGAGATTAATTTTGAAACCCGTGGAAAACGTGGCCAA   600NO: 7   529  GATGCGATGTATGAGTATATGGCTCAAGCCTGTGCAGGAAATCGTGTCAGGCGATCAGTA   588             ||||||||||||||| |||||||||||||||||||||||||||||||||||||||| |||NO: 8   601  GATGCGATGTATGAGTATATGGCTCAAGCCTGTGCAGGAAATCGTGTCAGGCGATCAGTA   660NO: 7   589  GGTAGCTCATTGTCATGCATCAACCTGGATTGGGATGTTATCCGTGATAAAACTAAAACT   648             ||||||||||||||| |||||||||||||||||||||||||||||||||||||||| |||NO: 8   661  GGTAGCTCATTGTCATGCATCAACCTGGATTGGGATGTTATCCGTGATAAAACTAAAACT   720NO: 7   649  AAGATCGAATCTCTGAAAGAACACGGTCCGATCAAAAACAAAATGAGCGAAAGCCCGAAC   708             ||||||||||||||| |||||||||||||||||||||||||||||||||||||||| |||NO: 8   721  AAGATCGAATCTCTGAAAGAACACGGTCCGATCAAAAACAAAATGAGCGAAAGCCCGAAC   780NO: 7   709  AAAACTGTATCTGAAGAAAAAGCTAAACAGTACCTGGAAGAATTCCACCAGACTGCACTG   768             ||||||||||||||| |||||||||||||||||||||||||||||||||||||||| |||NO: 8   781  AAAACTGTATCTGAAGAAAAAGCTAAACAGTACCTGGAAGAATTCCACCAGACTGCACTG   840NO: 7   769  GAACACCCGGAACTGTCTGAACTTAAGACCGTTACTGGTACCAACCCGGTATTCGCTGGT   828             ||||||||||||||| |||||||||||||||||||||||||||||||||||||||| |||NO: 8   841  GAACACCCGGAACTGTCTGAACTTAAGACCGTTACTGGTACCAACCCGGTATTCGCTGGT   900NO: 7   829  GCTAACTACGCTGCTTGGGCAGTAAACGTTGCTCAGGTTATCGATAGCGAAACTGCTGAT   888             ||||||||||||||| |||||||||||||||||||||||||||||||||||||||| |||NO: 8   901  GCTAACTACGCTGCTTGGGCAGTAAACGTTGCTCAGGTTATCGATAGCGAAACTGCTGAT   960NO: 7   889  AACCTGGAAAAAACTACCGCGGCTCTGTCTATCCTGCCGGGTATCGGTAGCGTAATGGGC   948             ||||||||||||||| |||||||||||||||||||||||||||||||||||||||| |||NO: 8   961  AACCTGGAAAAAACTACCGCGGCTCTGTCTATCCTGCCGGGTATCGGTAGCGTAATGGGC  1020NO: 7   949  ATCGCAGACGGCGCCGTTCACCACAACACTGAAGAAATCGTTGCACAGTCTATCGCTCTG  1008             ||||||||||||||| |||||||||||||||||||||||||||||||||||||||| |||NO: 8  1021  ATCGCAGACGGCGCCGTTCACCACAACACTGAAGAAATCGTTGCACAGTCTATCGCTCTG  1080NO: 7  1009  AGCTCTCTGATGGTTGCTCAGGCCATCCCGCTGGTAGGTGAACTGGTTGATATCGGTTTC  1068             ||||||||||||||| |||||||||||||||||||||||||||||||||||||||| |||NO: 8  1081  AGCTCTCTGATGGTTGCTCAGGCCATCCCGCTGGTAGGTGAACTGGTTGATATCGGTTTC  1140NO: 7  1069  GCTGCATACAACTTCGTTGAAAGCATCATCAACCTGTTCCAGGTTGTTCACAACTCTTAC  1128             ||||||||||||||| |||||||||||||||||||||||||||||||||||||||| |||NO: 8  1141  GCTGCATACAACTTCGTTGAAAGCATCATCAACCTGTTCCAGGTTGTTCACAACTCTTAC  1200NO: 7  1129  AACCGCCCGGCTTACTCTCCGGGTCACAAGACGCATGCACCTACTTCTAGCTCTACCAAG  1188             ||||||||||||||| |||||||||||||||||||||||||||||||||||||||| |||NO: 8  1201  AACCGCCCGGCTTACTCTCCGGGTCACAAGACGCATGCACCTACTTCTAGCTCTACCAAG  1260NO: 7  1189  AAAACCCAGCTGCAGCTCGAGCACCTGCTGCTGGATTTGCAGATGATCCTGAACGGTATC  1248             ||||||||||||||| |||||||||||||||||||||||||||||||||||||||| |||NO: 8  1261  AAAACCCAGCTGCAGCTCGAGCACCTGCTGCTGGATTTGCAGATGATCCTGAACGGTATC  1320NO: 7  1249  AACAATTACAAGAACCCGAAACTGACGCGTATGCTGACCTTCAAGTTCTACATGCCGAAG  1308             ||||||||||||||| |||||||||||||||||||||||||||||||||||||||| |||NO: 8  1321  AACAATTACAAGAACCCGAAACTGACGCGTATGCTGACCTTCAAGTTCTACATGCCGAAG  1380NO: 7  1309  AAGGCCACCGAACTGAAACACCTGCTGCAGTGTCTAGAAGAAGAACTGAAACCGCTGGAG  1368             ||||||||||||||| |||||||||||||||||||||||||||||||||||||||| |||NO: 8  1381  AAGGCCACCGAACTGAAACACCTGC---AGTGTCTAGAAGAAGAACTGAAACCGCTGGAG  1437NO: 7  1369  GAAGTTCTGAACCTGGCTCAGTCTAAAAACTTCCACCTGCGGCCGCGTGACCTGATCTCT  1428             ||||||||||||||| |||||||||||||||||||||||||||||||||||||||| |||NO: 8  1438  GAAGTTCTGAACCTGGCTCAGTCTAAAAACTTCCACCTGCGGCCGCGTGACCTGATCTCT  1497NO: 7  1429  AACATCAACGTAATCGTTCTGGAACTGAAGGGCTCTGAAACCACCTTCATGTGTGAATAC  1488             ||||||||||||||| |||||||||||||||||||||||||||||||||||||||| |||NO: 8  1498  AACATCAACGTAATCGTTCTGGAACTGAAGGGCTCTGAAACCACCTTCATGTGTGAATAC  1557NO: 7  1489  GCTGATGAGACCGCAACCATCGTAGAATTCCTGAACCGTTGGATCACCTTCTGTCAGTCT  1548             ||||||||||||||| |||||||||||||||||||||||||||||||||||||||| |||NO: 8  1558  GCTGATGAGACCGCAACCATCGTAGAATTCCTGAACCGTTGGATCACCTTCTGTCAGTCT  1617NO: 7  1549  ATCATCTCTACCCTGACC---                                         1566             ||||||||||||||| ||###NO: 8  1618  ATCATCTCTACCCTGACCTGA                                         1638(DNA sequence IL-2 portion of denileukin diftitox) SEQ ID NO: 9  1  GCACCTACTTCTAGCTCTACCAAGAAAACCCAGCTGCAGCTCGAGCACCT 51  GCTGCTGGATTTGCAGATGATCCTGAACGGTATCAACAATTACAAGAACC101  CGAAACTGACGCGTATGCTGACCTTCAAGTTCTACATGCCGAAGAAGGCC151  ACCGAACTGAAACACCTGCAGTGTCTAGAAGAAGAACTGAAACCGCTGGA201  GGAAGTTCTGAACCTGGCTCAGTCTAAAAACTTCCACCTGCGGCCGCGTG251  ACCTGATCTCTAACATCAACGTAATCGTTCTGGAACTGAAGGGCTCTGAA301  ACCACCTTCATGTGTGAATACGCTGATGAGACCGCAACCATCGTAGAATT351  CCTGAACCGTTGGATCACCTTCTGTCAGTCTATCATCTCTACCCTGACCT 401  GA < 402

Proteins Produced Using DNA Expression Vectors of the Present Invention

The first amino acid of a mature active diphtheria toxin related fusionprotein of the present invention is a glycine as shown in bold (aminoacid 1) in SEQ ID NOs: 13 and 15. The signal sequence within SEQ ID NO:4 is labeled with negative numbers, counting back from the first glycineof the mature fusion protein and has the following amino acid sequenceMSRKLFASILIGALLGIGAPPSAHA (SEQ ID NO: 22). The signal sequence is shownin SEQ ID NOs: 11 and 12 and is underlined. The mature secreteddiphtheria toxin fusion protein includes a diphtheria toxin portion,such as Gly₁-His₃₈₇, and a target receptor binding domain, such as anIL-2 protein from Ala388-Thr52o in SEQ ID NO: 3. Other target receptorbinding domains used in the present invention that may be fused to adiphtheria toxin protein (or functional part thereof) include IL-3,IL-4, TL-6, IL-7, IL-15, EGF, FGF, substance P, CD4, αMSH, GRP, TTfragment C, GCSF, heregulin β1, TNFα, TGFβ, among others, or acombination thereof. SEQ ID NO: 10 describes c-denileukin diftitox thatis not secreted and is requires purification from inclusion bodies in E.coli. SEQ ID NO: 12 describes immature secreted is-denileukm diftitoxwith a signal sequence. SEQ ID NO: 13 describes MS-denileukin diftitoxwherein the signal sequence has been cleaved off during the process ofsecretion to the extracellular space.

(Protein Sequence of c-denileukin diftitox known as Ontak ®)SEQ ID NO: 10   1  MGADDVVDSSKSFVMENFSSYHGTKP 27  GYVDSIQKGIQKPKSGTQGNYDDDWKGFYSTDNKYDAAGYSVDNENPLSG 77  KAGGVVKVTYPGLTKVLALKVDNAETIKKELGLSLTEPLMEQVGTEEFIK127  RFGDGASRVVLSLPFAEGSSSVEYINNWEQAKALSVELEINFETRGKRGQ177  DAMYEYMAQACAGNRVRRSVGSSLSCINLDWDVIRDKTKTKIESLKEHGP227  IKNKMSESPNKTVSEEKAKQYLEEFHQTALEHPELSELKTVTGTNPVFAG277  ANYAAWAVNVAQVIDSETADNLEKTTAALSILPGIGSVMGIADGAVHHNT327  EEIVAQSIALSSLMVAQAIPLVGELVDIGFAAYNFVESIINLFQVVHNSY377  NRPAYSPGHKTHAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTR427  MLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISN477  INVIVLELKCSETTFMCEYADETATIVEFLNRWITFCQSIISTLT < 521(w-diphtheria toxin) SEQ ID NO: 11  1  MSRKLFASILIGALLGIGAPPSAHAGADDVVDSSKSFVMENFSSYHGTKP 51  GYVDSIQKGIQKPKSGTQGNYDDDWKGFYSTDNKYDAAGYSVDNENPLSG101  KAGGVVKVTYPGLTKVLALKVDNAETIKKELGLSLTEPLMEQVGTEEFIK151  RFGDGASRVVLSLPFAEGSSSVEYINNWEQAKALSVELEINFETRGKRGQ201  DAMYEYMAQACAGNRVRRSVGSSLSCINLDWDVIRDKTKTKIESLKEHGP251  IKNKMSESPNKTVSEEKAKQYLEEFHQTALEHPELSELKTVTGTNPVFAG301  ANYAAWAVNVAQVIDSETADNLEKTTAALSILPGIGSVMGIADGAVHHNT351  EEIVAQSIALSSLMVAQAIPLVGELVDIGFAAYNFVESIINLFQVVHNSY401  NRPAYSPGHKTQPFLHDGYAVSWNTVEDSIIRTGFQGESGHDIKITAENT451  PLPIAGVLLPTIPGKLDVNKSKTHISVNGRKIRMRCRAIDGDVTFCRPKS501  PVYVONGVHANLHVAFHRSSSEKIHSNEISSDSIGVLGYQKTVDHTKVNS551  KLSLFFEIKS < 560 (is-denileukin diftitox) SEQ ID NO: 12-25  MSRKLFASILIGALLGIGAPPSAHA GADDVVDSSKSFVMENFSSYHGTKP 26  GYVDSIQKGIQKPKSGTQGNYDDDWKGFYSTDNKYDAAGYSVDNENPLSG 76  KAGGVVKVTYPGLTKVLALKVDNAETIKKELGLSLTEPLMEQVGTEEFIK126  RFGDGASRVVLSLPFAEGSSSVEYINNWEQAKALSVELEINFETRGKRGQ176  DAMYEYMAQACAGNRVRRSVGSSLSCINLDWDVIRDKTKTKIESLKEHGP226  IKNKMSESPNKTVSEEKAKQYLEEFHQTALEHPELSELKTVTGTNPVFAG276  ANYAAWAVNVAQVIDSETADNLEKTTAALSILPGIGSVMGIADGAVHHNT326  EEIVAQSIALSSLMVAQAIPLVGELVDIGFAAYNFVESIINLFQVVHNSY376  NRPAYSPGHKTHAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTR426  MLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISN476  INVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT < 520(ms-denileukin diftitox) SEQ ID NO: 13   1  GADDVVDSSKSFVMENFSSYHGTKP 26  GYVDSIQKGIQKPKSGTQGNYDDDWKGFYSTDNKYDAAGYSVDNENPLSG 76  KAGGVVKVTYPGLTKVLALKVDNAETIKKELGLSLTEPLMEQVGTEEFIK126  RFGDGASRVVLSLPFAEGSSSVEYINNWEQAKALSVELEINFETRGKRGQ176  DAMYEYMAQACAGNRVRRSVGSSLSCINLDWDVIRDKTKTKIESLKEHGP226  IKNKMSESPNKTVSEEKAKQYLEEFHQTALEHPELSELKTVTGTNPVFAG276  ANYAAWAVNVAQVIDSETADNLEKTTAALSILPGIGSVMGIADGAVHHNT326  EEIVAQSIALSSLMVAQAIPLVGELVDIGFAAYNFVESIINLFQVVHNSY376  NRPAYSPGHKTHAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTR426  MLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISN476  INVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT < 520(Protein sequence of is-denileukin diftitox-VLM) SEQ ID NO: 14-25  MSRKLFASILIGALLGIGAPPSAHA GADDVADSSKSFVMENFSSYHGTKP 26  GYVDSIQKGIQKPKSGTQGNYDDDWKGFYSTDNKYDAAGYSVDNENPLSG 76  KAGGVVKVTYPGLTKVLALKVDNAETIKKELGLSLTEPLMEQVGTEEFIK126  RFGDGASRVVLSLPFAEGSSSVEYINNWEQAKALSVELEINFETRGKRGQ176  DAMYEYMAQACAGNRVRRSVGSSLSCINLDWDVIRDKTKTKIESLKEHGP226  IKNKMSESPNKTVSEEKAKQYLEEFHQTALEHPELSELKTVTGTNPVFAG276  ANYAAWAVNVAQVIDSETADNLEKTTAALSILPGIGSVMGIADGAVHHNT326  EEIVAQSIALSSLMVAQAIPLVGELVDIGFAAYNFVESIINLFQVVHNSY376  NRPAYSPGHKTHAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTR426  MLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISN476  INVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT < 520(Protein sequence of ms-denileukin diftitox-VLM) SEQ ID NO: 15  1  GADDVADSSKSFVMENFSSYHGTKP 26  GYVDSIQKGIQKPKSGTQGNYDDDWKGFYSTDNKYDAAGYSVDNENPLSG 76  KAGGVVKVTYPGLTKVLALKVDNAETIKKELGLSLTEPLMEQVGTEEFIK126  RFGDGASRVVLSLPFAEGSSSVEYINNWEQAKALSVELEINFETRGKRGQ176  DAMYEYMAQACAGNRVRRSVGSSLSCINLDWDVIRDKTKTKIESLKEHGP226  IKNKMSESPNKTVSEEKAKQYLEEFHQTALEHPELSELKTVTGTNPVFAG276  ANYAAWAVNVAQVIDSETADNLEKTTAALSILPGIGSVMGIADGAVHHNT326  EEIVAQSIALSSLMVAQAIPLVGELVDIGFAAYNFVESIINLFQVVHNSY376  NRPAYSPGHKTHAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTR426  MLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISN476  INVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT < 520(Protein sequence of denileukin diftitox-VLM described in U.S. Pat. No. 8,865,866) SEQ ID NO: 16  1  MGADDVADSSKSFVMENFSSYHGTKP 27  GYVDSIQKGIQKPKSGTQGNYDDDWKGFYSTDNKYDAAGYSVDNENPLSG 77  KAGGVVKVTYPGLTKVLALKVDNAETIKKELGLSLTEPLMEQVGTEEFIK127  RFGDGASRVVLSLPFAEGSSSVEYINNWEQAKALSVELEINFETRGKRGQ177  DAMYEYMAQACAGNRVRRSVGSSLSCINLDWDVIRDKTKTKIESLKEHGP227  IKNKMSESPNKTVSEEKAKQYLEEFHQTALEHPELSELKTVTGTNPVFAG277  ANYAAWAVNVAQVIDSETADNLEKTTAALSILPGIGSVMGIADGAVHHNT327  EEIVAQSIALSSLMVAQAIPLVGELVDIGFAAYNFVESIINLFQVVHNSY377  NRPAYSPGHKTHAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTR427  MLTFKFYMPKKATELKHLLQCLEEELKPLEEVLNLAQSKNFHLRPRDLIS477  NINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT < 522

Protein Alignment of SEQ ID NO: 16 is denileukin diftitox-VLM describedin U.S. Pat. No. 8,865,866 that has an extra amino acid (L) at position445 when compared with SEQ ID NO: 14 is-denileukin diftitox-VLM of thepresent invention.

Similarity: 521/522 (99.81%)NO: 16    1  M------------------------GADDVADSSKSFVMENFSSYHGTKPGYVDSIQKGI   36             |########################||||||||||||||||||||||||||||||| ||| NO: 14    1  MSRKLFASILIGALLGIGAPPSAHAGADDVADSSKSFVMENFSSYHGTKPGYVDSIQKGI   60NO: 16   37  QKPKSGTQGNYDDDWKGFYSTDNKYDAAGYSVDNENPLSGKAGGVVKVTYPGLTKVLALK   96             ||||||||||||||| |||||||||||||||||||||||||||||||||||||||| |||NO: 14   61  QKPKSGTQGNYDDDWKGFYSTDNKYDAAGYSVDNENPLSGKAGGWKVTYPGLTKVLALK   120NO: 16   97  VDNAETIKKELGLSLTEPLMEQVGTEEFIKRFGDGASRVVLSLPFAEGSSSVEYINNWEQ  156             ||||||||||||||| |||||||||||||||||||||||||||||||||||||||| ||| NO: 14  121  VDNAETIKKELGLSLTEPLMEQVGTEEFIKRFGDGASRVVLSLPFAEGSSSVEYINNWEQ  180NO: 16  157  AKALSVELEINFETRGKRGQDAMYEYMAQACAGNRVRRSVGSSLSCINLDWDVIRDKTKT  216             ||||||||||||||| |||||||||||||||||||||||||||||||||||||||| |||NO: 14  181  AKALSVELEINFETRGKRGQDAMYEYMAQACAGNRVRRSVGSSLSCINLDWDVIRDKTKT  240NO: 16  217  KIESLKEHGPIKNKMSESPNKTVSEEKAKQYLEEFHQTALEHPELSELKTVTGTNPVFAG  276             ||||||||||||||| |||||||||||||||||||||||||||||||||||||||| |||NO: 14  241  KIESLKEHGPIKNKMSESPNKTVSEEKAKQYLEEFHQTALEHPELSELKTVTGTNPVFAG  300NO: 16  277  ANYAAWAVNVAQVIDSETADNLEKTTAALSILPGIGSVMGIABGAVHHNTEEIVAQSIAL  336             ||||||||||||||| |||||||||||||||||||||||||||||||||||||||| ||| NO: 14  301  ANYAAWAVNVAQVIDSETADNLEKTTAALSILPGIGSVMGIADGAVHHNTEEIVAQSIAL  360NO: 16  337  SSLMVAQAIPLVGELVDIGFAAYNFVESIINLFQWHNSYNRPAYSPGHKTHAPTSSSTK   396             ||||||||||||||| |||||||||||||||||||||||||||||||||||||||| ||| NO: 14  361  SSLMVAQAIPLVGELVDIGFAAYNFVESIINLFQWHNSYNRPAYSPGHKTHAPTSSSTK   420NO: 16  397  KTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLLQCLEEELKPLE  456             ||||||||||||||| ||||||||||||||||||||||||||||||||#||||||| ||| NO: 14  421  KTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHL-QCLEEELKPLE  479NO: 16  457  EVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQS  516             ||||||||||||||| |||||||||||||||||||||||||||||||||||||||| ||| NO: 14  480  EVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQS  539NO: 16  517  IISTLT                                                        522             |||||| NO: 14  540  IISTLT                                                        545

Use of DNA Expression Vectors to Manufacture Proteins.

The method using Fe-independent, secreted expression of proteins relatedto diphtheria toxin described above has several commercial applicationsin addition to the use of the method to express s-denileukin diftitox.The method can be used to improve (enhance) expression (yield) of:

WT Diphtheria Toxin:

The wild type Diphtheria toxin (SEQ ID NO: 11) used to make diphtheriatoxoid, a vaccine for diphtheria which is present in DTP, TDaP, andother combination vaccines may be made using the DNA expression vectorof the present invention. The DNA segment encoding SEQ ID NO: 11 may beplaced in the DNA expression vector of the present invention and locateddownstream of the ToxP/mutant ToxO.

Cross-Reacting Material-197 (CRM197) and Cross-Reacting Material-107(CRM107):

CRM197 and CR107 are mutant proteins of full-length diphtheria toxinwhich are highly immunogenic but are completely devoid of toxinactivity. They are used as carriers for several polysaccharide conjugatevaccines. For example, Wyeth and Pfizer took advantage of thisimmunogenicity in the 1990s when it conjugated seven polysaccharidesfrom Streptococcus pneumoniae to CRM197 to create the original Prevnarvaccine which was FDA approved in February 2000. A 13-polysaccharidesPrevnar was FDA-approved in 2010. The meningococcal vaccine Menveo, fromNovartis, is four Neissena meningitidis polysaccharides plus CRM197.This vaccine gained FDA approval in 2010. The cancer immunotherapycompany Imugene (ASX: IMU) reported dramatic improvements in antibodytiters from its B cell peptide cancer immunotherapy targeting HER2 whenit used CRM 197 as a carrier protein. CRM 197 is also being evaluated asa potential drug delivery protein. The Swiss-based TuringPharmaceuticals is working on CRM197 fusion constructs with therapeuticproteins of up to 1,000 amino acids in length. The DNA expressionvectors of the present invention maybe used to produce CRM 197 and CRM107. One or more of the DNA segment(s) encoding SEQ ID NOs: 18-21 may beplaced in the DNA expression vector of the present invention and locateddownstream of the ToxP/mutant ToxO.

Diphtheria Toxin Based Fusion Proteins with Cleavable Peptide or ProteinTags Used to Enhance Purification.

Cleavable peptide tags (such as His₆ (SEQ ID NO: 23) or FLAG [DYKDDDDK](SEQ ID NO: 24)) or protein tags (such as GST [glutathioneS-transferase] or SUMO [Small Ubiquitin-like Modifier protein]) may befused with specific protease cleavage sites to diphtheria toxin basedfusion proteins. Affinity chromatography methods using antibodies orligands which bind to the tag may be used for rapid purification of thetagged protein.

Following purification, the specific cleavage site enables separation ofthe tag from the desired diphtheria toxin related proteins. Such fusionsmay enhance purification of diphtheria toxin based fusion proteins ofthe present invention.

(Protein sequence of ms-CRM197) SEQ ID NO: 17  1  GADDVVDSSKSFVMENFSSYHGTKPGYVDSIQKGIQKPKSGTQGNYDDDW 51  KEFYSTDNKYDAAGYSVDNENPLSGKAGGVVKVTYPGLTKVLALKVDNAE101  TIKKELGLSLTEPLMEQVGTEEFIKRFGDGASRVVLSLPFAEGSSSVEYI151  NNWEQAKALSVELEINFETRGKRGQDAMYEYMAQACAGNRVRRSVGSSLS201  CINLDWDVIRDKTKTKIESLKEHGPIKNKMSESPNKTVSEEKAKQYLEEF251  HQTALEHPELSELKTVTGTNPVFAGANYAAWAVNVAQVIDSETADNLEKT301  TAALSILPGIGSVMGIADGAVHHNTEEIVAQSIALSSLMVAQAIPLVGEL351  VDIGFAAYNFVESIINLFQVVHNSYNRPAYSPGHKTQPFLHDGYAVSWNT401  VEDSIIRTGFQGESGHDIKITAENTPLPIAGVLLPTIPGKLDVNKSKTHI451  SVNGRKIRMRCRAIDGDVTFCRPKSPVYVGNGVHANLHVAFHRSSSEKIH501  SNEISSDSIGVLGYQKTVDHTKVNSKLSLFFEIKS < 535(Protein sequence of is-CRM197) SEQ ID NO: 18  1  MSRKLFASILIGALLGIGAPPSAHAGADDVVDSSKSFVMENFSSYHGTKP 51  GYVDSIQKGIQKPKSGTQGNYDDDWKEFYSTDNKYDAAGYSVDNENPLSG101  KAGGVVKVTYPGLTKVLALKVDNAETIKKELGLSLTEPLMEQVGTEEFIK151  RFGDGASRVVLSLPFAEGSSSVEYINNWEQAKALSVELEINFETRGKRGQ201  DAMYEYMAQACAGNRVRRSVGSSLSCINLDWDVIRDKTKTKIESLKEHGP251  IKNKMSESPNKTVSEEKAKQYLEEFHQTALEHPELSELKTVTGTNPVFAG301  ANYAAWAVNVAQVIDSETADNLEKTTAALSILPGIGSVMGIADGAVHHNT351  EEIVAQSIALSSLMVAQAIPLVGELVDIGFAAYNFVESIINLFQVVHNSY401  NRPAYSPGHKTQPFLHDGYAVSWNTVEDSIIRTGFQGESGHDIKITAENT451  PLPIAGVLLPTIPGKLDVNKSKTHISVNGRKIRMRCRAIDGDVTFCRPKS501  PVYVGNGVHANLHVAFHRSSSEKIHSNEISSDSIGVLGYQKTVDHTKVNS551  KLSLFFEIKS < 560 (Protein sequence of ms-CRM107) SEQ ID NO: 19                              GADDVVDSSKSFVMENFSSYHGTKP 51  GYVDSIQKGIQKPKSGTQGNYDDDWKGFYSTDNKYDAAGYSVDNENPLSG101  KAGGVVKVTYPGLTKVLALKVDNAETIKKELGLSLTEPLMEQVGTEEFIK151  RFGDGASRVVLSLPFAEGSSSVEYINNWEQAKALSVELEINFETRGKRGQ201  DAMYEYMAQACAGNRVRRSVGSSLSCINLDWDVIRDKTKTKIESLKEHGP251  IKNKMSESPNKTVSEEKAKQYLEEFHQTALEHPELSELKTVTGTNPVFAG301  ANYAAWAVNVAQVIDSETADNLEKTTAALSILPGIGSVMGIADGAVHHNT351  EEIVAQSIALSSLMVAQAIPLVGELVDIGFAAYNFVESIINLFQVVHNSY401  NRPAYSPGHKTQPFFHDGYAVSWNTVEDSIIRTGFQGESGHDIKITAENT451  PLPIAGVLLPTIPGKLDVNKSKTHISVNGRKIRMRCRAIDGDVTFCRPKS501  PVYVGNGVHANLHVAFHRSSSEKIHSNEISSDSIGVLGYQKTVDHTKVNF551  KLSLFFEIKS < 560 (Protein sequence of is-CRM107) SEQ ID NO: 20  1  MSRKLFASILIGALLGIGAPPSAHAGADDVVDSSKSFVMENFSSYHGTKP 51  GYVDSIQKGIQKPKSGTQGNYDDDWKGFYSTDNKYDAAGYSVDNENPLSG101  KAGGVVKVTYPGLTKVLALKVDNAETIKKELGLSLTEPLMEQVGTEEFIK151  RFGDGASRVVLSLPFAEGSSSVEYINNWEQAKALSVELEINFETRGKRGQ201  DAMYEYMAQACAGNRVRRSVGSSLSCINLDWDVIRDKTKTKIESLKEHGP251  IKNKMSESPNKTVSEEKAKQYLEEFHQTALEHPELSELKTVTGTNPVFAG301  ANYAAWAVNVAQVIDSETADNLEKTTAALSILPGIGSVMGIADGAVHHNT351  EEIVAQSIALSSLMVAQAIPLVGELVDIGFAAYNFVESIINLFQVVHNSY401  NRPAYSPGHKTQPFFHDGYAVSWNTVEDSIIRTGFQGESGHDIKITAENT451  PLPIAGVLLPTIPGKLDVNKSKTHISVNGRKIRMRCRAIDGDVTFCRPKS501  PVYVGNGVHANLHVAFHRSSSEKIHSNEISSDSIGVLGYQKTVDHTKVNF551  KLSLFFEIKS < 560

TABLE 1 SEQUENCE NUMBER DESCRIPTION SEQ ID NO: 38 Protein sequence of Nterminal His tag to VLM s-Ontak SEQ ID NO: 39 Protein sequence of Nterminal His tag to VLM s-Ontak after signal sequence is cleaved SEQ IDNO: 40 Protein sequence of N terminal His tag to VLM s-Ontak aftersignal sequence is cleaved and TEV site is cleaved SEQ ID NO: 41 DNAsequence of N terminal His tag to VLM s-Ontak SEQ ID NO: 42 Proteinsequence of C terminal His tag to VLM s-Ontak SEQ ID NO: 43 Proteinsequence of C terminal His tag to VLM s-Ontak after signal sequence iscleaved) SEQ ID NO: 44 DNA sequence of C terminal His tag to VLM s-OntakSEQ ID NO: 45 Protein sequence of C terminal TEV His9 tag to VLM s-Ontak(“His9” disclosed as SEQ ID NO: 48) SEQ ID NO: 46 Protein sequence of Cterminal TEV His9 tag to VLM s-Ontak after signal sequence is cleaved(“His9” disclosed as SEQ ID NO: 48) SEQ ID NO: 30 Protein sequence of Cterminal TEV His9 tag to VLM s-Ontak after signal sequence and Tevprotease site are cleaved (“His9” disclosed as SEQ ID NO: 48) SEQ ID NO:31 DNA sequence of C terminal His tag to VLM s-Ontak SEQ ID NO: 32Secreted C. diphtheriae protease 1 amino acid sequence SEQ ID NO: 33Secreted C. diphtheriae protease 1 DNA sequence SEQ ID NO: 34 DNAsequence of allelic exchange substrate [AES] for knocking out secretedC. diphtheriae protease 1 SEQ ID NO: 35 Secreted C. diphtheriae protease2 amino acid sequence SEQ ID NO: 36 Secreted C. diphtheriae protease 2DNA sequence) Protease 2 DNA sequence SEQ ID NO: 37 DNA sequence ofallelic exchange substrate [AES] for knocking out secreted C.diphtheriae protease 2Purification of VLM s-Ontak Using His-Tagged Versions of the Polypeptide

In some preparations of VLM s-Ontak produced in Corynebacteriumdiphtheriae C7 slow proteolytic cleavage of the mature 520 amino acidpolypeptide occurs. This is probably due to secreted proteases made byCorynebacterium diphtheriae C7. This proteolytic cleavage occurs atapproximately ammo acid 390 of the mature 520 ammo acid VLM s-Ontak.

Histidine-tagged (His-tagged) versions of VLM s-Ontak have beenconstructed for the purpose of accelerating the purification of thedesired protein away from the secreted proteases present in the culturesupernatant. Tobacco Etch Virus (TEV) nuclear-inclusion-a endopeptidase(EC 3.4.22.44) recognition sites have also been engineered into theseHis-tagged versions of VLM s-Ontak. The purpose of the TEV cleavagesites is to enable the removal of the poly-His sequences in the finalpreparation of VLM s-Ontak. TEV is a highly specific endopeptidase whichrecognizes the amino acid sequence ENLYFQ\X where ‘V denotes the cleavedpeptide bond, and X represents any small hydrophobic or polar amino acidsuch as glycine (G) (SEQ ID NO: 49).

N-terminal His-Tagged VLM s-Ontak with TEV Cleavage Site.

As shown in SEQ ID: 38 (Protein sequence of N terminal His tag to VLMs-Ontak) it is possible to add the amino sequence HHHHHHENLYFQ (SEQ IDNO: 50) to the immature protein sequence of VLM s-Ontak near itsN-termmus. In this version, the sequence HHHHHHENLYFQ (SEQ ID NO: 50)appears immediately after the 26 amino acid signal sequence andimmediately before the mature sequence of VLM s-Ontak (GADDVA (SEQ IDNO: 51)). The first glycine of VLM s-Ontak comprises the finalrecognition residue for the TEV protease which recognizes ENLYFQ\X (SEQID NO: 49) with X being any small amino acid. The mature, secretedprotein sequence of this N-terminal His-tagged VLM s-Ontak is shown inSEQ ID: 39 (Protein sequence of N terminal His tag to VLM s-Ontak aftersignal sequence is cleaved) which is a good candidate for Nickel-columnaffinity purification with its His₆ tag (SEQ ID NO: 23). The affinitypurified VLM s-Ontak may then be exposed to small amounts of pure TEVprotease, leading to enzymatic proteolysis that removes the 13N-terminal residues MHHHHHHENLYFQ (SEQ ID NO: 52) and releases mature,untagged VLM s-Ontak as is shown in SEQ ID NO: 40 (Protein sequence of Nterminal His tag to VLM s-Ontak after signal sequence is cleaved and TEVsite is cleaved).

Because the secreted protease(s) of Corynebacterium diphtheriae C7cleave at approximately amino acid 390, N-terminal His-tagging can leadto two species: full length desired VLM s-Ontak (520 amino acids) and a390-amino acid N-termmal breakdown fragment. These two polypeptides,being relatively close in size (as well as molecular composition) aredifficult to separate by size exclusion chromatography. Hence we havealso developed C-terminal His-tagged version of VLM s-Ontak.

C-Terminal His-Tagged VLM s-Ontak Without TEV Cleavage Wite.

As shown in SEQ ID NO: 42 (Protein sequence of C terminal His tag to VLMs-Ontak) it is possible to add the amino sequence HHHHHH (SEQ ID NO: 23)to the immature protein sequence of VLM s-Ontak at its C-terminus. Inthis version, the sequence HHHHHH (SEQ ID NO: 23) appears immediatelyafter the C-termmal threonine of VLM s-Ontak ( . . . IISTLT (SEQ ID NO:53)). The mature, secreted protein sequence of this C-terminalHis-tagged VLM s-Ontak is shown in SEQ ID: 43 (Protein sequence of Cterminal His tag to VLM s-Ontak after signal sequence is cleaved) whichis a good candidate for Nickel-column affinity purification with itsHis₆ tag (SEQ ID NO: 23).

C-Terminal His-Tagged VLM s-Ontak with TEV Cleavage Site.

In order to avoid having the His₆ sequence (SEQ ID NO: 23) in the finalpolypeptide sequence of the above version of VLM s-Ontak made byC-terminal His-tagging (SEQ ID: 43), it is possible to insert a TEVrecognition sequence at the C-terminus to enable removal of the His-tagsequence. In this version, the sequence ENLYFQGHHHHHHHHH (SEQ ID NO: 54)appears immediately after the C-terminal threonine of VLM s-Ontak ( . .. IISTLT (SEQ ID NO: 53)). Since nickel affinity binding is enhanced bypoly-His sequences even longer than six amino acids, it is possible toinclude nine His residues. The amino acid sequence of this C-terminalHis-tagged VLM s-Ontak with TEV cleavage site is shown in SEQ ID: 45(Protein sequence of C terminal TEV His9 tag (SEQ ID NO: 48) to VLMs-Ontak). The mature, secreted protein sequence of this C-terminalHis-tagged VLM s-Ontak with TEV cleavage site is shown in SEQ ID: 46(Protein sequence of C terminal TEV His9 tag (SEQ ID NO: 48) to VLMs-Ontak after signal sequence is cleaved) and is a good candidate forNickel-column affinity purification with its His? tag (SEQ ID NO: 48).The affinity purified VLM s-Ontak may then be exposed to small amountsof pure TEV protease, leading to enzymatic proteolysis that removes the10 C-terminal residues GHHHHHHHHH (SEQ ID NO: 55), and releases mature,untagged VLM s-Ontak as is shown in SEQ ID: 30. Of note, this version ofpurified VLM s-Ontak (SEQ ID: 30) is 526 amino acids in length ratherthan 520 ammo acids (SEQ ID NO: 15) because it contains six additionalamino acids of the TEV protease recognition sequence (ENLYFQ (SEQ ID NO:56) fused to the usual C-terminus threonine of VLM s-Ontak ( . . .IISTLT (SEQ ID NO: 53)). The end result of this version of C-terminalHis-tagged VLM s-Ontak with TEV cleavage site (SEQ ID: 30) is aC-terminal sequence . . . IISTLTENLYFQ (SEQ ID NO: 57).

Manufacturing Method for VLM s-Ontak Which Include His-Tags and TEVProtease Sites.

The above three His-tag versions of VLM s-Ontak (N-terminal His₆ tag(SEQ ID NO: 23) with TEV protease site, C-terminal His₆ tag (SEQ ID NO:23) without TEV protease site, and C-terminal His9 (SEQ ID NO: 48) tagwith TEV protease site) are examples of methods to use His-tag/Nickelcolumn affinity chromatography in the manufacturing method of VLMs-Ontak. Because of secreted proteases from Corynebacterium diphtheriaeC7 that are present in the culture supernatant, it is important topurify VLM s-Ontak away from other proteins in the culture supernatantrapidly in order to avoid significant loss of the desired product. Theinclusion of His-tags and TEV protease sites represents a significantimprovement and may enable a rapid, streamlined manufacturing processfor VLM s-Ontak.

Generation of Corynebacterium diphtheriae C7 Lacking Key SecretedProteases for Improved Manufacturing of VLM s-Ontak.

The genome sequence of Corynebacterium diphtheriae C7 reveals twosecreted proteases: Protease 1 is NCBI Reference Sequence WP_014318592.1(SEQ ID: 32, 33) and Protease 2 is NCBI Reference Sequence WP014318898.1 (SEQ ID: 35, 36). These proteases may be genetically deletedusing the method of Ton-That and Schneewind (Ton-That H, Schneewind O.Assembly of pili on the surface of Corynebacterium diphtheriae. MolMicrobiol. 2003 Nov;50(4): 1429-38. PubMed PMID: 14622427) and alsoAllen and Schmitt (Allen C E, Schmitt M P. HtaA is an iron-regulatedhemin binding protein involved in the utilization of heme iron inCorynebacterium diphtheriae. J Bacteriol. 2009 Apr;191(8):2638-48.PubMed PMID: 19201805). The allelic exchange substrates to knock outprotease 1 and protease 2 are shown in SEQ ID: 34 and SEQ ID: 37,respectively. These sequences when inserted into pk18mobsacB, aconjugative, mating plasmid with sacB counterselection (Schafer A, TauchA, Jäger W, Kalinowski J, Thierbach G, Pühler A (1994) Small mobilizablemulti-purpose cloning vectors derived from the Escherichia coli plasmidspK18 and pK19: selection of defined deletions in the chromosome ofCorynebacterium glutumicum. Gene 145:69-73. PMID: 8045426), lead toconstructs which will knockout each protease. A recombinantCorynebacterium diphtheriae strain lacking both protease 1 and protease2 will be a valuable production strain for future manufacturing methodsto generate VLM s-Ontak.

Protein Manufacturing Process of Diphtheria Toxin-Based Fusion Proteins

Using the DNA plasmids and expression vectors of the present invention,a novel process was discovered eliminating the problems associated withthe conventional method of manufacturing Ontak®. Ontak® is currentlyexpressed using a DNA vector in an E. coli expression system,c-denileukin diftitox or Ontak® is 521 amino acids in length and has amolecular weight of 58 kD. The conventional Ontak® manufacturing processresults in the formation of Ontak® aggregates of heterogeneous molecularweight, residual DNA, and excessive residual detergent in the finalformulation resulting in the FDA placing classic-Ontak® on clinical holdin June 2011. As observed in FIG. 8a , Ontak® is expressed from aplasmid in E. coli and results in insoluble, cytosolic Ontak® (protein)accumulations known as inclusion body forms. Using the process of thepresent invention, FIG. 8b illustrates the expression of s-denileukmdiftitox as an extracellular mature secreted protein in a cell freesupernatant that can be easily purified and results in higher proteinyields as illustrated in FIG. 9. FIG. 9 shows both a Coomassie Bluestain for total protein and an anti-IL2 immunoblot of s-demleukindiftitox generated using the process of the present invention probedwith anti-IL-2.

The novel process of the present invention comprises: 1) transformingbacteria, preferably a Corynebacterium diphtheria strain, with a DNAexpression vector of the present invention, 2) forming a transformant;3) incubating the transformant in a culture medium for a period of rimeto allow growth and expression of a protein (such as a diphtheriatoxin-based fusion protein and CRM typically containing a signalpeptide), 4) secretion of the protein into the culture medium (due to asignal peptide attached to the protein); and (8) purifying thediphtheria toxin-based fusion protein from the culture medium. The DNAexpression vectors include a ToxP and mutant ToxO that regulate theexpression of at least one protein, such as a diphtheria toxin fusionprotein, CRM protein, or other protein that may be attached to a signalpeptide of the present invention.

Therapeutic Applications of Diphtheria Toxin-Based Fusion Proteins ofthe Present Invention

Clinical efficacy of Ontak® has been demonstrated in cutaneous T celllymphoma, peripheral T cell lymphoma, steroid-refractory graft versushost disease, methotrexate-refractory psoriasis, andmethotrexate-refractory rheumatoid arthritis. Clinical efficacy has alsobeen demonstrated in malignant melanoma and ovarian carcinoma as shownin FIG. 14. The diphtheria toxin-based fusion proteins of the presentinvention (including s-denileukin diftitox, ms-denileukin diftitox,is-denileukin diftitox-VLM, ms-denileukin diftitox-VLM) produced by themethods of the present invention will perform similarly, or better, thanOntak® that is commercially available with regard to clinical efficaciesof treating or preventing disease.

Treatment for Tuberculosis.

As illustrated in FIG. 10, inventors of the present invention believediphtheria toxin fusion proteins of the present invention will be activeagainst tuberculosis. Denileukin diftitox is known to depleteIL-2-receptor (CD25+)-bearing cells including T regulatory (T_(regs))cells. T_(regs) cells express CD25 as well as FoxP3 and areimmunosuppressive by their inhibition of Teffector (T_(cff)) cells.T_(cff) cells such as CD4+Thelper (T_(h)) cells and CD8+cytotoxic Tlymphocytes (CTLs) are needed within a tuberculosis granuloma to containthe M. tuberculosis bacterial infection. During tuberculosis infection,cellular lesions called granulomas form to contain the infection but areunable to fully eradicate the bacilli. Regulatory T cells (Tregs) arerecruited to granulomas, leading to suppression of effector T cellfunction, potentially contributing to a permissive environment for M.tuberculosis persistence and growth. The diphtheria toxin fusionproteins of the present invention are used to deplete Tregs, whichexpress IL-2 receptor, in order to ameliorate immune suppression bythese cells during TB infection. FIG. 11 illustrates diphtheria fusionproteins used in the in vivo treatment of subjects (mice) with M.tuberculosis. Mice were infected with M. tb. strain H37R_(v) by aerosolinfection giving an initial implantation of ˜2.8 log₁₀ CFU counts inlungs on day 0. The groups of mice were treated with 750 ng of c-Ontak®intraperitoneally (IP) or intravenously (IV) as one treatment cycle (1×,dosed at week 2 post-infection) or two treatment cycles (2×, dosed at˜day 3 pre-infection and week 2 post-infection). A treatment cycle ofdenileukin diftitox is defined as 35 mg/kg (750 ng for a typical mouse)given two times, two days apart. RHZ daily treatment by oral gavage wasstarted at week 2. R is rifampin and was given to mice at 10 mg/kg. H isisoniazid and was given to mice at 10 mg/kg. Z is pyrazinamide and wasgiven to mice at 150 mg/kg. The outcome of this study is illustrated inFIGS. 12 and 13.

Treatment for Cancer

Tregs have also been shown to inhibit anti-tumor immunity, and thecellular expansion of Tregs in tumors generally correlates with poorprognosis in patients. Denileukin diftitox treatment in melanomapatients resulted in transient depletion of Tregs and increased 1 yearmedian overall survival, s-denileukm diftitox and s-denileukindiftitox-VLM of the present invention will be used to deplete Tregs inpatients with tumors heavily infiltrated with Tregs as a cancerimmunotherapy.

Nucleic Acid and Protein Sequences of s-Ontak-His₆ (“His₆” Disclosed asSEQ ID NO: 23)

Protein Sequence of C. diphtherias derived s-Ontak-His₆ (“His₆”disclosed as SEQ ID NO: 23) (theoretical MW 58339) (IL2 portion inBoldface) (SEQ ID NO: 58):

                         GADDVVDSSKSFVMENFSSYHGTKPGYVDSIQKGIQKPKSGTQGNYDDDWKGFYSTDNKYDAAGYSVDNENPLSGKAGGVVKVTYPGLTKVLALKVDNAETIKKELGLSLTEPLMEQVGTEEFIKRFGDGASRVVLSLPFAEGSSSVEYINNWEQAKALSVELEINFETRGKRGQDAMYEYMAQACAGNRVRRSVGSSLSCINLDWDVIRDKTKTKIESLKEHGPIKNKMSESPNKTVSEEKAKQYLEEFHQTALEHPELSELKTVTGTNPVFAGANYAAWAVNVAQVIDSETADNLEKTTAALSILPGIGSVMGIADGAVHHNTEEIVAQSIALSSLMVAQAIPLVGELVDIGFAAYNFVESIINLFQVVHNSYNRPAYSPGHKTHAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLTHHHHH H

DNA sequence for C. diphtheriae-derived s-Ontak-His₆ (“His₆” disclosedas SEQ ID NO: 23) (SEQ ID NO: 59). Alterations to promoter/operator arestarred with boldface. The underlined portion encodes the signalsequence. The first codon of mature s-Ontak-His₆ (“His₆” disclosed asSEQ ID NO: 23) begins at base 149 in larger font and italicized. Thecodons for the C-terminal His₆ (“His₆” disclosed as SEQ ID NO: 23) beginat base 1709 in larger font and italicized.

                                        **** * *   1  TTGATTTCAGAGCACCCTTATAATTAGGATAGCTAAGTCCATTA       TTTTAT  51  GAGTCCTGGTAAGGGGATACGTTGTGAGCAGAAAACTGTTTGCG       TCAATC 101  TTAATAGGGGCGCTACTGGGGATAGGGGCCCCACCTTCAGCCCA       TGCAGG 151  CGCTGATGATGTTGTTGATTCTTCTAAATCTTTTGTGATGGAAA       ACTTTT 201  CTTCGTACCACGGGACTAAACCTGGTTATGTAGATTCCATTCAA       AAAGGT 251  ATACAAAAGCCAAAATCTGGTACACAAGGAAATTATGACGATGA       TTGGAA 301  AGGGTTTTATAGTACCGACAATAAATACGACGCTGCGGGATACT       CTGTAG 351  ATAATGAAAACCCGCTCTCTGGAAAAGCTGGAGGCGTGGTCAAA       GTGACG 401  TATCCAGGACTGACGAAGGTTCTCGCACTAAAAGTGGATAATGC       CGAAAC 451  TATTAAGAAAGAGTTAGGTTTAAGTCTCACTGAACCGTTGATGG       AGCAAG 501  TCGGAACGGAAGAGTTTATCAAAAGGTTCGGTGATGGTGCTTCG       CGTGTA 551  GTGCTCAGCCTTCCCTTCGCTGAGGGGAGTTCTAGCGTTGAATA       TATTAA 601  TAACTGGGAACAGGCGAAAGCGTTAAGCGTAGAACTTGAGATTA       ATTTTG 651  AAACCCGTGGAAAACGTGGCCAAGATGCGATGTATGAGTATATG       GCTCAA 701  GCCTGTGCAGGAAATCGTGTCAGGCGATCAGTAGGTAGCTCATT       GTCATG 751  CATCAACCTGGATTGGGATGTTATCCGTGATAAAACTAAAACTA       AGATCG 801  AATCTCTGAAAGAACACGGTCCGATCAAAAACAAAATGAGCGAA       AGCCCG 851  AACAAAACTGTATCTGAAGAAAAAGCTAAACAGTACCTGGAAGA       ATTCCA 901  CCAGACTGCACTGGAACACCCGGAACTGTCTGAACTTAAGACCG       TTACTG 951  GTACCAACCCGGTATTCGCTGGTGCTAACTACGCTGCTTGGGCA       GTAAAC1001  GTTGCTCAGGTTATCGATAGCGAAACTGCTGATAACCTGGAAAA       AACTAC1051  CGCGGCTCTGTCTATCCTGCCGGGTATCGGTAGCGTAATGGGCA       TCGCAG1101  ACGGCGCCGTTCACCACAACACTGAAGAAATCGTTGCACAGTCT       ATCGCT1151  CTGAGCTCTCTGATGGTTGCTCAGGCCATCCCGCTGGTAGGTGA       ACTGGT1201  TGATATCGGTTTCGCTGCATACAACTTCGTTGAAAGCATCATCA       ACCTGT1251  TCCAGGTTGTTCACAACTCTTACAACCGCCCGGCTTACTCTCCG       GGTCAC1301  AAGACGCATGCACCTACTTCTAGCTCTACCAAGAAAACCCAGCT       GCAGCT1351  CGAGCACCTGCTGCTGGATTTGCAGATGATCCTGAACGGTATCA       ACAATT1401  ACAAGAACCCGAAACTGACGCGTATGCTGACCTTCAAGTTCTAC       ATGCCG1451  AAGAAGGCCACCGAACTGAAACACCTGCAGTGTCTAGAAGAAGA       ACTGAA1501  ACCGCTGGAGGAAGTTCTGAACCTGGCTCAGTCTAAAAACTTCC       ACCTGC1551  GGCCGCGTGACCTGATCTCTAACATCAACGTAATCGTTCTGGAA       CTGAAG1601  GGCTCTGAAACCACCTTCATGTGTGAATACGCTGATGAGACCGC       AACCAT1651  CGTAGAATTCCTGAACCGTTGGATCACCTTCTGTCAGTCTATCA       TCTCTA1701  CCCTGACCCACCATCACCATCATCACTGA < 1711

All references, including publications, patent applications, andpatents, cited herein are hereby incorporated by reference to the sameextent as if each reference were individually and specifically indicatedto be incorporated by reference and were set forth in its entiretyherein.

The use of the terms “a” and “an” and “the” and similar referents in thecontext of describing the invention (especially in the context of thefollowing claims) are to be construed to cover both the singular and theplural, unless otherwise indicated herein or clearly contradicted bycontext. The terms “comprising,” “having,” “including,” and “containing”are to be construed as open-ended terms (i.e., meaning “including, butnot limited to,”) unless otherwise noted. Recitation of ranges of valuesherein are merely intended to serve as a shorthand method of referringindividually to each separate value falling within the range, unlessotherwise indicated herein, and each separate value is incorporated intothe specification as if it were individually recited herein. All methodsdescribed herein can be performed in any suitable order unless otherwiseindicated herein or otherwise clearly contradicted by context. The useof any and all examples, or exemplary language (e.g., “such as”)provided herein, is intended merely to better illuminate the inventionand does not pose a limitation on the scope of the invention unlessotherwise claimed. No language in the specification should be construedas indicating any non-claimed element as essential to the practice ofthe invention.

Preferred embodiments of this invention are described herein, includingthe best mode for carrying out the invention. Variations of thosepreferred embodiments may become apparent to those of ordinary skill inthe art upon reading the foregoing description. The inventors expectskilled artisans to employ such variations as appropriate, and theinventors intend for the invention to be practiced otherwise than asspecifically described herein. Accordingly, this invention includes allmodifications and equivalents of the subject matter recited in theclaims appended hereto as permitted by applicable law. Moreover, anycombination of the described elements of the invention in all possiblevariations thereof is encompassed by the invention unless otherwiseindicated herein or otherwise clearly contradicted by context.

Embodiments of the disclosure concern methods and/or compositions fortreating and/or preventing disorders such as cancer and tuberculosis inwhich a subject is administered a composition of the present inventioncomprising a nucleic acid or protein sequence such as anyone of SEQ IDNOs: 11-15, or fusion proteins thereof

An individual known to having disease such as cancer and/ortuberculosis, suspected of having such a disease, or at risk for havingsuch a disease may be provided an effective amount of a composition ofthe present invention comprising a nucleic acid or protein sequence suchas anyone of SEQ ID NOs: 11-15, or fusion proteins thereof. Those atrisk for cancer or tuberculosis may be those individuals having one ormore genetic factors, may be of advancing age, and/or may have a familyhistory, for example.

In particular embodiments of the disclosure, an individual is given anagent for cancer and/or tuberculosis therapy in addition to acomposition of the present invention comprising a nucleic acid orprotein sequence such as anyone of SEQ ID NOs: 11-15, or fusion proteinsthereof. Such additional therapy may include chemotherapy orantimicrobial agents, for example. When combination therapy is employedwith a composition of the present invention comprising a nucleic acid orprotein sequence such as anyone of SEQ ID NOs: 11-15, or fusion proteinsthereof, the additional therapy may be given prior to, at the same timeas, and/or subsequent to a composition of the present inventioncomprising a nucleic acid or protein sequence such as anyone of SEQ IDNOs: 11-15, or fusion proteins thereof.

Pharmaceutical Preparations

Pharmaceutical compositions of the present invention comprise aneffective amount of one or more composition of the present inventioncomprising a nucleic acid or protein sequence such as anyone of SEQ IDNOs: 11-15, or fusion proteins thereof, dissolved or dispersed in apharmaceutically acceptable carrier. The phrases “pharmaceutical orpharmacologically acceptable” refers to molecular entities andcompositions that do not produce an adverse, allergic or other untowardreaction when administered to an animal, such as, for example, a human,as appropriate. The preparation of a pharmaceutical composition thatcomprises at least one composition of the present invention comprising anucleic acid or protein sequence such as any one of SEQ ID NOs: 11-15,or fusion proteins thereof, or additional active ingredient will beknown to those of skill in the art in light of the present disclosure,as exemplified by Remington: The Science and Practice of Pharmacy,21^(st) Ed. Lippincott Williams and Wilkins, 2005, incorporated hereinby reference. Moreover, for animal (e.g., human) administration, it willbe understood that preparations should meet sterility, pyrogenicity ,general safety and purity standards as required by FDA Office ofBiological Standards.

As used herein, “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, surfactants, antioxidants,preservatives (e.g., antibacterial agents, antifungal agents), isotonicagents, absorption delaying agents, salts, preservatives, drugs, drugstabilizers, gels, binders, excipients, disintegration agents,lubricants, sweetening agents, flavoring agents, dyes, such likematerials and combinations thereof, as would be known to one of ordinaryskill in the art (see, for example, Remington's Pharmaceutical Sciences,18th Ed. Mack Printing Company, 1990, pp. 1289-1329, incorporated hereinby reference). Except insofar as any conventional carrier isincompatible with the active ingredient, its use in the pharmaceuticalcompositions is contemplated.

The one or more compositions of the present invention comprising anucleic acid or protein sequence such as anyone of SEQ ID NOs: 11-15, orfusion proteins thereof, may comprise different types of carriersdepending on whether it is to be administered in solid, liquid oraerosol form, and whether it need to be sterile for such routes ofadministration as injection. The present compositions can beadministered intravenously, intradermally, transdermally, intrathecally,intraarterially, intraperitoneally, intranasally, intravaginally,intrarectally, topically, intramuscularly, subcutaneously, mucosally,orally, topically, locally, inhalation (e.g., aerosol inhalation),injection, infusion, continuous infusion, localized perfusion bathingtarget cells directly, via a catheter, via a lavage, in cremes, in lipidcompositions (e.g., liposomes), or by other method or any combination ofthe forgoing as would be known to one of ordinary’ skill in the art(see, for example, Remington's Pharmaceutical Sciences, 18th Ed. MackPrinting Company, 1990, incorporated herein by reference).

The one or more compositions of the present invention comprising anucleic acid or protein sequence such as any one of SEQ ID NOs: 11-15.or fusion proteins thereof, may be formulated into a composition in afree base, neutral or salt form. Pharmaceutically acceptable salts,include the acid addition salts, e.g., those formed with the free aminogroups of a proteinaceous composition, or which are formed withinorganic acids such as for example, hydrochloric or phosphoric acids,or such organic acids as acetic, oxalic, tartaric or mandelic acid.Salts formed with the free carboxyl groups can also be derived frominorganic bases such as for example, sodium, potassium, ammonium,calcium or ferric hydroxides; or such organic bases as isopropylamine,trimethylamine, histidine or procaine. Upon formulation, solutions willbe administered in a manner compatible with the dosage formulation andin such amount as is therapeutically effective. The formulations areeasily administered in a variety of dosage forms such as formulated forparenteral administrations such as injectable solutions, or aerosols fordelivery to the lungs, or formulated for alimentary administrations suchas drug release capsules and the like.

Further in accordance with the present disclosure, the composition ofthe present invention suitable for administration is provided in apharmaceutically acceptable carrier with or without an inert diluent.The carrier should be assimilable and includes liquid, semi-solid, i.e.,pastes, or solid carriers. Except insofar as any conventional media,agent, diluent or carrier is detrimental to the recipient or to thetherapeutic effectiveness of a composition contained therein, its use inadministrable composition for use in practicing the methods of thepresent invention is appropriate. Examples of carriers or diluentsinclude fats, oils, water, saline solutions, lipids, liposomes, resins,binders, fillers and the like, or combinations thereof. The compositionmay also comprise various antioxidants to retard oxidation of one ormore component. Additionally, the prevention of the action ofmicroorganisms can be brought about by preservatives such as variousantibacterial and antifungal agents, including but not limited toparabens (e.g., methylparabens, propylparabens), chlorobutanol, phenol,sorbic acid, thimerosal or combinations thereof.

In accordance with the present invention, the composition is combinedwith the carrier in any convenient and practical manner, i.e., bysolution, suspension, emulsification, admixture, encapsulation,absorption and the like. Such procedures are routine for those skilledin the art.

In a specific embodiment of the present invention, the composition iscombined or mixed thoroughly with a semi-solid or solid earner. Themixing can be earned out in any convenient manner such as grinding.Stabilizing agents can be also added in the mixing process in order toprotect the composition from loss of therapeutic activity, i.e.,denaturation in the stomach. Examples of stabilizers for use in an thecomposition include buffers, amino acids such as glycine and lysine,carbohydrates such as dextrose, mannose, galactose, fructose, lactose,sucrose, maltose, sorbitol, mannitol, etc.

In further embodiments, the present invention may concern the use of apharmaceutical lipid vehicle composition that includes one or morecomposition of the present invention comprising a nucleic acid orprotein sequence such as anyone of SEQ ID NOs: 11-15, or fusion proteinsthereof, one or more lipids, and an aqueous solvent. As used herein, theterm “lipid” will be defined to include any of a broad range ofsubstances that is characteristically insoluble in water and extractablewith an organic solvent. This broad class of compounds are well known tothose of skill in the art, and as the term “lipid” is used herein, it isnot limited to any particular structure. Examples include compoundswhich contain long-chain aliphatic hydrocarbons and their derivatives. Alipid may be naturally occurring or synthetic (i.e., designed orproduced by man). However, a lipid is usually a biological substance.Biological lipids are well known in the art, and include for example,neutral fats, phospholipids, phosphoglycerides, steroids, terpenes,lysolipids, glycosphingolipids, glycolipids, sulphatides, lipids withether and ester-linked fatty’ acids and polymerizable lipids, andcombinations thereof. Of course, compounds other than those specificallydescribed herein that are understood by one of skill in the art aslipids are also encompassed by the compositions and methods of thepresent invention.

One of ordinary skill in the art would be familiar with the range oftechniques that can be employed for dispersing a composition in a lipidvehicle. For example, the one or more compositions of the presentinvention comprising a nucleic acid or protein sequence such as anyoneof SEQ ID NOs: 11-15, or fusion proteins thereof may be dispersed in asolution containing a lipid, dissolved with a lipid, emulsified with alipid, mixed with a lipid, combined with a lipid, covalently bonded to alipid, contained as a suspension in a lipid, contained or complexed witha micelle or liposome, or otherwise associated with a lipid or lipidstructure by any means known to those of ordinary skill in the art. Thedispersion may or may not result in the formation of liposomes.

The actual dosage amount of a composition of the present inventionadministered to an animal patient can be determined by physical andphysiological factors such as bodyweight, severity of condition, thetype of disease being treated, previous or concurrent therapeuticinterventions, idiopathy of the patient and on the route ofadministration. Depending upon the dosage and the route ofadministration, the number of administrations of a preferred dosageand/or an effective amount may vary according to the response of thesubject. The practitioner responsible for administration will, in anyevent, determine the concentration of active ingredient(s) in acomposition and appropriate dose(s) for the individual subject.

In certain embodiments, pharmaceutical compositions may comprise, forexample, at least about 0.1% of an active compound. In otherembodiments, an active compound may comprise between about 2% to about75% of the weight of the unit, or between about 25% to about 60%, forexample, and any range derivable therein. Naturally, the amount ofactive compound(s) in each therapeutically useful composition may beprepared is such a way that a suitable dosage will be obtained in anygiven unit dose of the compound. Factors such as solubility,bioavailability, biological half-life, route of administration, productshelf life, as well as other pharmacological considerations will becontemplated by one skilled in the art of preparing such pharmaceuticalformulations, and as such, a variety of dosages and treatment regimensmay be desirable.

In other non-limiting examples, a dose may also comprise from about 1microgram/kg/body weight, about 5 microgram/kg/body weight, about 10microgram/kg/body weight, about 50 microgram/kg/body weight, about 100microgram/kg/body weight, about 200 microgram/kg/body weight, about 350microgram/kg/body weight, about 500 microgram/kg/body weight, about 1milligram/kg/body weight, about 5 milligram/kg/body weight, about 10milligram/kg/body weight, about 50 milligram/kg/body weight, about 100milligram/kg/body weight, about 200 milligram/kg/body weight, about 350milligram/kg/body weight, about 500 milligram/kg/body weight, to about1000 mg/kg/body weight or more per administration, and any rangederivable therein. In non-limiting examples of a derivable range fromthe numbers listed herein, a range of about 5 mg/kg/body weight to about100 mg/kg/body weight, about 5 microgram/kg/body weight to about 500milligram/kg/body weight, etc., can be administered, based on thenumbers described above.

In further embodiments, a pharmaceutical composition of the invention asdescribed in any of the previous embodiments comprises greater thanabout 80% purity of a poly peptide of the invention. In otherembodiments, the pharmaceutical composition comprises greater than about81%, greater than about 82%, greater than about 83%, greater than about84%, greater than about 85%, greater than about 86%, greater than about87%, greater than about 88%, greater than about 89%, greater than about90%, greater than about 91%, greater than about 92%, greater than about93%, greater than about 94%, greater than about 95%, greater than about95%, greater than about 96%, greater than about 97%, greater than about98%, greater than about 99%, or about 100% purity of a polypeptide ofthe invention. In other embodiments, the pharmaceutical compositioncomprises about 80%, about 81%, about 82%, about 83%, about 84%, about85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%,about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about98%, about 99%, or about 100% purity of a polypeptide of the invention.In other embodiments, the pharmaceutical composition comprises fromabout 80% to about 100%, from about 80% to about 97%, from about 80% toabout 95%, from about 80% to about 90%, from about 80% to about 85%,from about 85% to about 100%, from about 85% to about 97%, from about85% to about 95%, from about 85% to about 90%, from about 90% to about100%, from about 90% to about 97%, from about 90% to about 95%, fromabout 95% to about 100%, or from about 95% to about 97% purity of apolypeptide of the invention, or any other range thereof.

In further embodiments, a pharmaceutical composition of the invention asdescribed in any of the previous embodiments comprises greater thanabout 80% aggregate-free, full-length, monomeric polypeptide of theinvention. In other embodiments, the pharmaceutical compositioncomprises greater than about 81%, greater than about 82%, greater thanabout 83%, greater than about 84%, greater than about 85%, greater thanabout 86%, greater than about 87%, greater than about 88%, greater thanabout 89%, greater than about 90%, greater than about 91%, greater thanabout 92%, greater than about 93%, greater than about 94%, greater thanabout 95%, greater than about 95%, greater than about 96%, greater thanabout 97%, greater than about 98%, greater than about 99%, or about 100%aggregate-free, full-length, monomeric polypeptide of the invention. Inother embodiments, the pharmaceutical composition comprises about 80%,about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%,about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, orabout 100% aggregate-free, full-length, monomeric polypeptide of theinvention. In other embodiments, the pharmaceutical compositioncomprises from about 80% to about 100%, from about 80% to about 97%,from about 80% to about 95%, from about 80% to about 90%, from about 80%to about 85%, from about 85% to about 100%, from about 85% to about 97%,from about 85% to about 95%, from about 85% to about 90%, from about 90%to about 100%, from about 90% to about 97%, from about 90% to about 95%,from about 95% to about 100%, or from about 95% to about 97%aggregate-free, full-length, monomeric polypeptide of the invention, orany other range thereof.

In further embodiments, a pharmaceutical composition of the inventioncomprises greater than about 80% purity of a polypeptide of theinvention (or any other range or amount described herein) and greaterthan about 80% aggregate-free, full-length, monomeric polypeptide of theinvention (or any other range or amount described herein).

In further embodiments, a polypeptide of such pharmaceuticalcompositions comprises a histidine (His) tag. In some embodiments, theHis tag has six or nine His residues. In other embodiments, the His tagis at the C-terminus of the polypeptide. In other embodiments, apolypeptide of such pharmaceutical compositions does not comprise a Histag.

Alimentary Compositions and Formulations

In one embodiment of the present disclosure, the one or morecompositions of the present invention comprising a nucleic acid orprotein sequence such as anyone of SEQ ID NOs: 11-15, or fusion proteinsthereof, are formulated to be administered via an alimentary’ route.Alimentary routes include all possible routes of administration in whichthe composition is in direct contact with the alimentary tract.Specifically, the pharmaceutical compositions disclosed herein may beadministered orally, buccally, rectally, or sublingually. As such, thesecompositions may be formulated with an inert diluent or with anassimilable edible earner, or they may be enclosed in hard- orsoft-shell gelatin capsule, or they may be compressed into tablets, orthey may be incorporated directly with the food of the diet.

In certain embodiments, the active compounds may be incorporated withexcipients and used in the form of ingestible tablets, buccal tables,troches, capsules, elixirs, suspensions, syrups, wafers, and the like(Mathiowitz et al., 1997; Hwang et al., 1998; U.S. Pat. Nos. 5,641,515;5,580,579 and 5,792,451, each specifically incorporated herein byreference in its entirety). The tablets, troches, pills, capsules andthe like may also contain the following: a binder, such as, for example,gum tragacanth, acacia, cornstarch, gelatin or combinations thereof; anexcipient, such as, for example, dicalcium phosphate, mannitol, lactose,starch, magnesium stearate, sodium saccharine, cellulose, magnesiumcarbonate or combinations thereof; a disintegrating agent, such as, forexample, com starch, potato starch, alginic acid or combinationsthereof; a lubricant, such as, for example, magnesium stearate; asweetening agent, such as, for example, sucrose, lactose, saccharin orcombinations thereof; a flavoring agent, such as, for examplepeppermint, oil of Wintergreen, cherry flavoring, orange flavoring, etc.When the dosage unit form is a capsule, it may contain, in addition tomaterials of the above type, a liquid carrier. Various other materialsmay be present as coatings or to otherwise modify the physical form ofthe dosage unit. For instance, tablets, pills, or capsules may be coatedwith shellac, sugar, or both. When the dosage form is a capsule, it maycontain, in addition to materials of the above type, carriers such as aliquid carrier Gelatin capsules, tablets, or pills may be entericallycoated. Enteric coatings prevent denaturation of the composition in thestomach or upper bowel where the pH is acidic. See, e.g., U.S. Pat. No.5,629,001. Upon reaching the small intestines, the basic pH thereindissolves the coating and permits the composition to be released andabsorbed by specialized cells, e.g., epithelial enterocytes and Peyer'spatch M cells. A syrup or elixir may contain the active compound sucroseas a sweetening agent methyl and propylparabens as preservatives, a dyeand flavoring, such as cherry or orange flavor. Of course, any materialused in preparing any dosage unit form should be pharmaceutically pureand substantially non-toxic in the amounts employed. In addition, theactive compounds may be incorporated into sustained-release preparationand formulations.

For oral administration the compositions of the present disclosure mayalternatively be incorporated with one or more excipients in the form ofa mouthwash, dentifrice, buccal tablet, oral spray, or sublingualorally-administered formulation. For example, a mouthwash may beprepared incorporating the active ingredient in the required amount inan appropriate solvent, such as a sodium borate solution (Dobell'sSolution). Alternatively, the active ingredient may be incorporated intoan oral solution such as one containing sodium borate, glycerin andpotassium bicarbonate, or dispersed in a dentifrice, or added in atherapeutically-effective amount to a composition that may includewater, binders, abrasives, flavoring agents, foaming agents, andhumectants. Alternatively the compositions may be fashioned into atablet or solution form that may be placed under the tongue or otherwisedissolved in the mouth.

Additional formulations which are suitable for other modes of alimentaryadministration include suppositories. Suppositories are solid dosageforms of various weights and shapes, usually medicated, for insertioninto the rectum. After insertion, suppositories soften, melt or dissolvein the cavity fluids. In general, for suppositories, traditionalcarriers may include, for example, polyalkylene glycols, triglyceridesor combinations thereof. In certain embodiments, suppositories may beformed from mixtures containing, for example, the active ingredient inthe range of about 0.5% to about 10%, and preferably about 1% to about2%.

Parenteral Compositions and Formulations

In further embodiments, one or more composition of the present inventioncomprising a nucleic acid or protein sequence such as anyone of SEQ IDNOs: 11-15, or fusion proteins thereof, may be administered via aparenteral route. As used herein, the term “parenteral” includes routesthat bypass the alimentary tract. Specifically, the pharmaceuticalcompositions disclosed herein may be administered for example, but notlimited to intravenously, intradermally, intramuscularly,intraarterially, intrathecally, subcutaneous, or intraperitoneally U.S.Pat. Nos. 6,7537,514; 6,613,308; 5,466,468; 5,543,158; 5,641,515; and5,399,363 (each specifically incorporated herein by reference in itsentirety).

Solutions of the active compounds as free base or pharmacologicallyacceptable salts may be prepared in water suitably mixed with asurfactant, such as hydroxypropylcellulose. Dispersions may also beprepared in glycerol, liquid polyethylene glycols, and mixtures thereofand in oils. Under ordinary’ conditions of storage and use, thesepreparations contain a preservative to prevent the growth ofmicroorganisms. The pharmaceutical forms suitable for injectable useinclude sterile aqueous solutions or dispersions and sterile powders forthe extemporaneous preparation of sterile injectable solutions ordispersions (U.S. Pat. 5,466,468, specifically incorporated herein byreference in its entirety) Tn all cases the form must be sterile andmust be fluid to the extent that easy injectability exists. It must bestable under the conditions of manufacture and storage and must bepreserved against the contaminating action of microorganisms, such asbacteria and fungi. The carrier can be a solvent or dispersion mediumcontaining, for example, water, ethanol, polyol (i.e., glycerol,propylene glycol, and liquid polyethylene glycol, and the like),suitable mixtures thereof, and/or vegetable oils. Proper fluidity may bemaintained, for example, by the use of a coating, such as lecithin, bythe maintenance of the required particle size in the case of dispersionand by the use of surfactants. The prevention of the action ofmicroorganisms can be brought about by various antibacterial andantifungal agents, for example, parabens, chlorobutanol, phenol, sorbicacid, thimerosal, and the like. In many cases, it will be preferable toinclude isotonic agents, for example, sugars or sodium chloride.Prolonged absorption of the injectable compositions can be brought aboutby the use in the compositions of agents delaying absorption, forexample, aluminum monostearate and gelatin.

For parenteral administration in an aqueous solution, for example, thesolution should be suitably buffered if necessary’ and the liquiddiluent first rendered isotonic with sufficient saline or glucose. Theseparticular aqueous solutions are especially suitable for intravenous,intramuscular, subcutaneous, and intraperitoneal administration. In thisconnection, sterile aqueous media that can be employed will be known tothose of skill in the art in light of the present disclosure. Forexample, one dosage may be dissolved in isotonic NaCl solution andeither added hypodermoclysis fluid or injected at the proposed site ofinfusion, (see for example, “Remington's Pharmaceutical Sciences” 15thEdition, pages 1035-1038 and 1570-1580). Some variation in dosage willnecessarily occur depending on the condition of the subject beingtreated. The person responsible for administration will, in any event,determine the appropriate dose for the individual subject. Moreover, forhuman administration, preparations should meet sterility, pyrogenicity,general safety and purity standards as required by FDA Office ofBiologics standards.

Sterile injectable solutions are prepared by incorporating the activecompounds in the required amount in the appropriate solvent with variousof the other ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle which contains the basic dispersion medium and the requiredother ingredients from those enumerated above. In the case of sterilepowders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum-drying and freeze-dryingtechniques which yield a pow der of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof A powdered composition is combined with a liquidcarrier such as, e.g., water or a saline solution, with or without astabilizing agent.

Miscellaneous Pharmaceutical Compositions and Formulations

In other preferred embodiments of the invention, the one or morecompositions of the present invention comprising a nucleic acid orprotein sequence such as anyone of SEQ ID NOs: 11-15, or fusion proteinsthereof, may be formulated for administration via various miscellaneousroutes, for example, topical (i.e., transdermal) administration, mucosaladministration (intranasal, vaginal, etc.) and/or inhalation.

Pharmaceutical compositions for topical administration may include theactive compound formulated for a medicated application such as anointment, paste, cream or powder. Ointments include all oleaginous,adsorption, emulsion and water-soluble based compositions for topicalapplication, while creams and lotions are those compositions thatinclude an emulsion base only. Topically administered medications maycontain a penetration enhancer to facilitate adsorption of the activeingredients through the skin. Suitable penetration enhancers includeglycerin, alcohols, alkyl methyl sulfoxides, pyrrolidones andluarocapram. Possible bases for compositions for topical applicationinclude polyethylene glycol, lanolin, cold cream and petrolatum as wellas any other suitable absorption, emulsion or water-soluble ointmentbase. Topical preparations may also include emulsifiers, gelling agents,and antimicrobial preservatives as necessary to preserve the activeingredient and provide for a homogenous mixture. Transdermaladministration of the present invention may also comprise the use of a“patch”. For example, the patch may supply one or more active substancesat a predetermined rate and in a continuous manner over a fixed periodof time.

In certain embodiments, the pharmaceutical compositions may be deliveredby eye drops, intranasal sprays, inhalation, and/or other aerosoldelivery vehicles. Methods for delivering compositions directly to thelungs via nasal aerosol sprays has been described e.g., in U.S. Pat.Nos. 5,756,353 and 5,804,212 (each specifically incorporated herein byreference in its entirety). Likewise, the delivery of drugs usingintranasal microparticle resins (Takenaga et al., 1998) andlysophosphatidyl-glycerol compounds (U.S. Pat. No. 5,725,871,specifically incorporated herein by reference in its entirety) are alsowell-known in the pharmaceutical arts. Likewise, transmucosal drugdelivery in the form of a polytetrafluoroetheylene support matrix isdescribed in U.S. Pat. No. 5,780,045 (specifically incorporated hereinby reference in its entirety).

The term aerosol refers to a colloidal system of finely divided solid ofliquid particles dispersed in a liquefied or pressurized gas propellant.The typical aerosol of the present invention for inhalation will consistof a suspension of active ingredients in liquid propellant or a mixtureof liquid propellant and a suitable solvent. A Suitable propellantsinclude hydrocarbons and hydrocarbon ethers. Suitable containers willvary according to the pressure requirements of the propellant.Administration of the aerosol will vary according to subject's age,weight and the severity and response of the symptoms.

Kits of the Disclosure

Any of the compositions described herein may be comprised in a kit. In anon-limiting example, one or more composition of the present inventioncomprising a nucleic acid or protein sequence such as anyone of SEQ IDNOs: 11-15, or fusion proteins thereof, may be comprised in a kit.

The kits may comprise a suitably aliquoted of one or more compositionsof the present invention comprising a nucleic acid or protein sequencesuch as anyone of SEQ ID NOs: 11-15, or fusion proteins thereof, and insome cases, one or more additional agents. The component(s) of the kitsmay be packaged either in aqueous media or in lyophilized form. Thecontainer means of the kits wall generally include at least one vial,test tube, flask, bottle, syringe or other container means, into which acomponent may be placed, and preferably, suitably aliquoted. Where thereare more than one component in the kit, the kit also will generallycontain a second, third or other additional container into which theadditional components may be separately placed. However, variouscombinations of components may be comprised in a vial. The kits of thepresent invention also will typically include a means for containing theone or more compositions of the present invention comprising a nucleicacid or protein sequence such as anyone of SEQ ID NOs: 11-15, or fusionproteins thereof, and any other reagent containers in close confinementfor commercial sale. Such containers may include injection orblow-molded plastic containers into which the desired vials areretained.

When the components of the kit are provided in one and/or more liquidsolutions, the liquid solution is an aqueous solution, with a sterileaqueous solution being particularly preferred. The one or morecompositions of the present invention comprising a nucleic acid orprotein sequence such as anyone of SEQ ID NOs: 11-15, or fusion proteinsthereof, may be formulated into a syringeable composition. In whichcase, the container means may itself be a syringe, pipette, and/or othersuch like apparatus, from which the formulation may be applied to aninfected area of the body, injected into an animal, and/or even appliedto and/or mixed with the other components of the kit.

However, the components of the kit may be provided as dried powder(s).When reagents and/or components are provided as a dry powder, the powdercan be reconstituted by the addition of a suitable solvent. It isenvisioned that the solvent may also be provided in another containermeans.

Although the invention has been described with reference to thepresently preferred embodiment, it should be understood that variousmodifications can be made without departing from the spirit of theinvention. Accordingly, the invention is limited only by the followingclaims.

What is claimed is:
 1. A method for producing aggregate-free,full-length, monomeric diphtheria toxin fusion proteins comprising: a)transforming bacteria with a DNA expression vector comprising a toxP, amutant toxO that blocks Fe-mediated regulation of gene expression, and aDNA sequence encoding a protein, wherein the toxP and the mutant toxOregulate expression of the DNA sequence encoding the protein; b)incubating the transformed bacteria in a culture medium to allowexpression of the protein that is secreted into the culture medium; andc) purifying the protein from the culture medium.
 2. The method of claim1, wherein the bacteria is Corynebacterium diphtheria.
 3. A method forproducing aggregate-free, full-length, monomeric diphtheria toxin fusionproteins comprising: a) transforming Corynebacterium diphtheria strainwith a DNA vector comprising: i) a toxP; ii) a mutant toxO that blocksFe-mediated regulation of gene expression; iii) a DNA sequence encodinga protein comprising: a) signal peptide; b) diphtheria receptor bindingdomain or has a non-functional diphtheria toxin receptor binding domain,and c) a target receptor binding domain selected from the groupcomprising IL-2, IL-3, IL-4, IL-6, IL-7, IL-15, EGF, FGF, substance P,CD4, αMSH, GRP, TT fragment C, GCSF, heregulin β1, TNFα, TGFβ, afunctional part thereof, or a combination thereof, wherein the toxP andthe mutant toxO regulate expression of the DNA sequence encoding theprotein; b) incubating the transformed bacteria in a culture medium toallow expression of the protein and that is secreted into the culturemedium; and c) purifying the diphtheria toxin fusion protein from theculture medium.
 4. The method of claim 3, wherein the diphtheria toxinreceptor fusion protein is selected from the any one of SEQ ID NOs:12 toSEQ ID NO:15.
 5. The method of claim 3, wherein the Corynebacteriumdiphtheria strain is Corynebacterium C7 beta (−), tox (−).
 6. A methodof making a protein comprising: a) providing a DNA expression vectorcomprising a toxP, a mutant toxO that blocks Fe-mediated regulation ofgene expression, a signal sequence, and a DNA sequence encoding aprotein; b) transforming a bacteria strain with the DNA vector to form atransformant; c) incubating the transformant in a culture medium for aperiod of time to allow expression of a protein that is secreted intothe culture medium; and d) purifying the protein from the culturemedium.